Aquaculture Food and Marketing

Economic development in West Virginia is critical, particularly in rural communities where traditional economic activities (principally coal and timber) have declined. Rural economic development has been a focus of the Aquaculture Food and Marketing Development Project (AFMDP). Strategies where aquaculture can impact economic development in West Virginia and adjacent states are: (1) development of mine water resources for commercial production of fish to eat, and (2) use of farm raised fish in recreation. Benefits derived from the work proposed in this document have potential for farmers growing fish for both food and recreation. As such the work will encourage development of a dual market for growers in the region strengthening both components of the industry. Work presented in this proposal is designed to complement the work which has been completed and presently underway.

1. Mine Water Aquaculture Productivity through Year-Round Operation with Recreationally Relevant Species Investigate the productivity of hybrid striped bass, largemouth bass, hybrid bluegill and a heat tolerant strain of rainbow trout in treated mine water to assess feasibility of year-round use of treated mine water.

2. Development of Cost Competetive HFRP Fish Tanks. To significantly reduce costs of HFRP for raceway systems by introducing recycled FRP products as partial substitution of chop-strand mats currently being used for the manufacture of tanks installed in WV. The goal is to combine the easy-assembly design being developed in Grant 6 with lower recycled material costs (Grant 8) to produce HFRP tanks at comparable prices with concrete.

3. Software tool for simulation of a flowing water raceway system. Develop and verify a software tool designed to simulate potential performance of a flowing water raceway system.

5. Protein and Lipid Recovery from Fish Processing By-products The objective is to scale up the protein and lipid recovery batch process to continuous semi-industrial application.

6. Technology Transfer. Information developed from this research and previous research will be integrated into an effort to educate people regarding how aquaculture is conducted and its role in economic development.

Much of the research supported by the Aquaculture Food and Marketing Development Project has involved production and processing of trout. A long term objective is to develop a modular raceway system and improve raceway design for commercial production of food fish in flowing water systems utilizing impaired water.

Development of Honeycomb Fiber Reinforced Polymer Tanks for Field Assembly from Flat Panels .

Several designs to join two flat panels were proposed and studied and the model considered to be best suitable for the current application was adopted for the new connector design. This design utilized a 4x4x4 angle plate with ¼” thickness and 3 bolts, 2 with Φ ¼”, normal head and one with Φ ¾”, sunken head, to join the side panel to the bottom panel. The ¾” bolt also has a threaded hole so that one of the ¼” bolts can be inserted into it. Holes are drilled into the flat panels corresponding to the location of the bolts and the angle plate is placed on the bottom panel. The ¾” bolt is inserted through the angle plate into the bottom panel with the threaded hole in the longitudinal direction of the core. The side panel is placed at one end and is bolted to the angle plate. The other ¼” bolt is then inserted through the side panel into the bottom panel and is tightened into the threaded hole in the ¾” bolt, to form a watertight connection. An elastomeric pad is used between the side panel and the bottom panel to provide an even surface of contact

Based on formulations proposed by Davalos and Chen (2005), the coefficient of elastic restraint was calculated and finite element models were created. Two sets of prototype connections were assembled, one with a 2” bottom panel and the other with a 4” bottom panel. On assembly of the first set of samples, it was decided that modifications to improve stiffness should be implemented before testing the connection. Experimental testing for stiffness and strength of the connection were performed and modified finite element models were created and are discussed in the later sections.

Modified design. The sunken head bolt was replaced with a regular bolt and the thickness of the angle plate was increased from 1/8” to ¼” to increase stiffness. Also, two 3/8” bolts were used to attach the angle plate to the side panel instead of one ¼” bolt. The ¼” bolt which is inserted into the ¾” bolt was also replaced with a 3/8” bolt of the same length. An adhesive sealant was used between the angle plate and the FRP panels to provide perfect contact between the two. A 1/8” steel plate was used as a washer on the outside of the side panel for the 3/8” bolts. Using these components, we assembled two prototype connectors, one with a 2” thick bottom panel and the other with a 4” thick bottom panel.

Experimental Testing of Connection Samples. The two connections assembled were tested first in the linear range and then to failure, to determine the rotational stiffness and strength of the connection. The experimental test setup was similar to the setup used to test the original stiffened and un-stiffened connection samples. The bottom panel is clamped rigidly to a vertical steel column. The side panel rests on a 4” wide support and has an overhanging length of 42”. As in the previous tests, the side panel is loaded at a distance of 36” from the joint and deflections at 24” and 36” are noted. Five strain gages are bonded to the side panel, to record the strain distribution near the joint.

Finite Element Modeling of the Connection. Solid elements are used for the FE analysis of the connection samples for ease of modeling. The side and bottom HFRP panels are modeled to the required dimensions using equivalent properties. A connection assembly is separately modeled as a single entity as shown in Figure 2.

The mesh that is generated for all the components and elements corresponding to the holes on the FRP panels are then deleted. Contact surfaces are created where surface interaction is expected, such as simulating contact between the side and bottom panels. It is also assumed that the sample is resting over 1” thick plate, as observed in the lab, which allows for angular rotation of the joint between the angle plate and the panels. Nuts of the corresponding bolts are tied to the nodes on top of them, simulating the actual experimental condition, where the nuts are in compression and hence can be assumed to be fixed to the panels. Boundary and loading conditions are applied to the model similar to the experimental setup.

Conclusion. By observing the failure of the samples on both experimental testing and finite element modeling, it can be seen that local failure of the material occurs around the washer of the ¾” bolt, for both the two panel thicknesses used. This may be avoided by using a larger washer, which distributes the stress over a larger area, thus increasing the strength of the connection. It can also be seen that the angle plate separates from the bottom panel as the load increases, which permits the washer to cut into the material. This separation of the angle plate from the panels allows for rotation of the connection (θ). Using a bigger washer would solve this problem to some extent, by resisting the separation forces. Thus from the experimental testing and finite element modeling, it can be observed that the 4” thick bottom panel is stiffer and stronger than the 2” thick bottom panel and hence is more suitable for the modified connection. The new connection design developed in this project offers the potential for economic field assembly of raceway systems using flat panels and relatively simple connections designs.

Production evaluation of two commercial diets for rainbow trout in treated mine water. An experiment comparing two commercially available diets will be completed in May, 2005. The control diet is a commonly used fish meal based diet and the second diet is a a commercially available diet made without fish meal. The diets will be assessed in terms of the following: (1) productivity related factors such as feed conversion ratio, growth rate, and survival, etc., (2) quantity and characteristics of solid and dissolved waste production, and (3) concentrations of organochlorine compounds (e.g., polychlorinated biphenyls, dioxins, etc.) in fish flesh grown in WVU’s mine water raceway.

Due to the potential impacts these findings may have on the aquaculture industry in WV, it is important to determine whether a difference in organochlorine body burdens can be detected between fish raised on a fish meal based diet and those grown using an all vegetable diet. In addition to fish production related benefits, vegetable-based feeds are reported to decrease solids and dissolved waste production, as a greater fraction of the total feed is utilized efficiently by the fish. This work is currently ongoing.

Conduct economic analyses to evaluate costs and benefits of using impaired water for the production of fish for food and recreation. As a precursor to determining profitability of mine water aquaculture, a data base consisting of all high-flow mine sites (>1000gpm) was updated. Measurements included measured flows, county location, land-owner, water quality data, treatment on site, power availability, and a production-related risk rating. We have completed an assessment of profitability (from an aquaculture producer’s perspective) and potential state-wide economic development impacts as a result of mine water aquaculture.

Develop designs for increasing the efficiency for removal of solid wastes from the quiescent zone in raceway systems producing trout. Solids removal mechanisms in flow-through aquaculture systems generally consist of quiescent settling in a chamber followed by evacuation through a standpipe, vacuuming, etc. These methods are known to be time and labor intensive and are generally inefficient. Prior to attempting to design and model more efficient quiescent zones and solids removal systems, it is necessary to first establish baseline hydraulic operating characteristics in conventional systems. Acoustic Doppler velocimetry (ADV) was used for the in situ measurement of three-dimensional velocities at discrete locations in flowing fluids, based on the Doppler Principle. In this work, hydrodynamic properties in a typical raceway system and quiescent zone were characterized using ADV technology and conclusions relevant to improving overall operation and future modifications to removal processes in raceways were developed.

Development and verification of a model to describe production of rainbow trout in raceway systems. Work to date on this project has focused on developing Excel-based software to design and/or evaluate an existing raceway. Currently, the software performs calculations for a single tank in the raceway system using user-defined inputs. These inputs include: seasonal water temperature, initial fish size, desired final size of fish, water flow rate, raceway dimensions, nutrient density of the diet, etc. Using accepted growth rate figures and other information (Soderberg 1995, Klontz 1991, and Westers 2003), the growth and optimal feeding histories are calculated along with oxygen usage and nitrogen generation rates within the tank.

Characterize the impact of varying CO₂ and O₂ levels on growth efficiency, nutrient utilization, and fillet attributes of Rainbow Trout and Arctic Charr. Rainbow trout (Oncorhynchus mykiss) grown in water with elevated free CO₂ display lethargic and intermittent feeding behavior, and have slower growth rates compared to fish grown in water with CO₂ concentrations < 25 mg/L. Reasons for the decreased growth are not yet known; however, increased activity levels (i.e during feeding) may result in transient acidosis and reduced oxygen consumption. If the reduced growth of hypercapnic rainbow trout is associated with transient acidosis and reduced respiratory function, then minimizing oxygen demands and activity levels associated with feeding may improve their growth. Feeding smaller meals throughout the day, instead of feeding one large meal in the morning, may help reduce activity levels associated with feeding and subsequent acidosis.

Two strains of rainbow trout (approximately 150 g per fish) were randomly stocked into twenty-four circular, 100-L tanks at a rate of 10 fish per tank. Carbon dioxide and feed frequency treatments were initiated at the beginning of the acclimation period. Fish were acclimated for 14 days to allow for adjustment to the new tank and treatment conditions. Carbon dioxide concentrations and water flows were adjusted as needed to obtain treatment CO₂ concentrations and maintain oxygen levels greater than 80% saturation.

Fish were acclimated to either 20 or 40 mg/L free CO₂ levels. In addition, two control tanks (without fish) were used. Each tank was equipped with transparent, removable polypropylene lids to minimize gas exchange between the water-air interface (Figure 3). The lids have small, snug-fitting holes for passage of the water and gas lines, as well as unsealed hole for feed dispersal and escape of CO₂ gas that might otherwise accumulate. Tanks were fitted with lids and gas lines before stocking the fish. Each CO₂ treatment level was fed one of two feeding frequencies during the acclimation period; either once per day or four times per day. Fish were fed 5% body weight per day (as either one feeding or spread over 4 feedings). Treatment combinations were in triplicate.

On the day of the experiment, CO₂ and feed treatments were continued as during the acclimation period. Feed intake was monitored throughout the 24-h experiment: out-flowing stand-pipes were equipped with netting to catch any uneaten feed. Oxygen and total ammonia-N (TAN) measurements were obtained from the inlet water and out-flowing stand-pipe using handheld meters and standard water quality test kits (YSI, Inc. and Hach Co.). Oxygen and TAN measurements were used for determination of oxygen consumption and nitrogen excretion rates. Carbon dioxide, pH, total alkalinity and total hardness concentrations were measured in the out-flowing stand-pipe using standard test kits (Hach Co.) and standard water quality methods.

Preliminary results in Figure 2 indicate that fish fed once per day (9 AM) had maximum oxygen consumptions earlier (near noon sample) compared to the fish fed throughout the day (9 AM, 11 AM, 1 PM, and 3 PM).

Figure 3. Legend: carbon dioxide level - times fed per day - strain of fish.

Omega-3 Fortified Rainbow Trout The four month feeding trial has been completed at the Reymann Memorial Farm. Fillets recovered from the trout fed a diet with 0, 15, and 23.5% of flaxseed oil were analyzed for moisture, total fat and vitamin E contents as well as fatty acid profile and lipid oxidation. Fillets were also in a storage stability study at 2 and 10°C. The fillets were analyzed for moisture, total fat and vitamin E contents as well as fatty acid profile and lipid oxidation. Preliminary data are presented in figure 4. Omega 3 fatty acid content per gram of flesh on a dry weight basis was 23.9, 17.18, and 46.4 for respective flaxseed oil treatments of 0, 15, and 23.5% at the end of the experiment. The omega 3 to omega 6 ratio was 1.98, 1.81, and 2.41, respectively. This study has been completed and the data analysis is in progress.

Cryprotection and Restructured Fish. Cryoprotectants other than sucrose/sorbitol were evaluated to reduce the sweetness of restructured trout products during frozen storage. Bacterial growth, lipid oxidation, thaw loss, cook yield, color, and texture were evaluated after 1 d, and 3 and 6 m of storage at -20 °C. Sucrose/sorbitol, trehalose, and trehalose/sorbitol, at 8% equally exhibited a cryoprotective action, minimized thaw loss and texture changes, while sodium lactate did not at 2% during 6-m frozen storage. Raw, carbohydrate-treated products had less L* values (Lightness as measured with a Minolta chromameter ) than the control and sodium lactate ones. Following cooking, no difference in L* value was observed. Cryoprotectants and frozen storage time did not affect bacterial growth and lipid oxidation of raw products.

Use of Near Infrared Spectroscopy to predict, real-time, texture development in trout products The purpose of this study was to determine the relationship between NIR spectra and storage modulus (G’, the elastic component) of low-fat beef or trout batters, with or without NaCl. Skinned rainbow trout fillets or unpeeled beef knuckles (IMPS #167) were minced and formulated to contain 10% fat, 30% added water, and either 0 or 2% NaCl. Batters were stuffed into the molds and cooked to 72 °C in water bath. Gels were removed when the internal temperature reached 50, 60, and 72 °C. The NIR spectra between 800 to 1700 nm were collected on these gels. The dynamic rheological property of the batter was performed on a rheometer at 50-Pa stress and 0.1-Hz frequency. The batter was heated from 10 to 72 °C at a temperature ramp of 1 °C/min, and the rheological property was expressed in terms of the storage modulus. Partial least squares (PLS) analysis was used to predict the storage modulus from NIR spectra of the gels from 96 data sets. A PLS cross-validation model used log (1/R) and log (G’). The experiment was conducted with 8 replications. The optimal model conditions for PLS regression occurred with 6 PLS factors at SEC = 0.56 and r = 0.89 (n = 64). When the PLS model was tested against the validation subset, similar performance was obtained at SEP = 0.52 and r = 0.91 (n = 32). During thermal processing, NIR can be used to predict the storage modulus of low-fat meat batters prepared from muscle of two species and at differing salt levels. Application of NIR spectroscopy to texture assessment during thermal processing will facilitate designing muscle foods for consumers with varying needs.

Vitamin E Stability During Fillet Storage and Handling. Fillets were processed from trout fed a diet containing either 200 (a commercial diet) or 5000 (a vitamin E supplemented diet) mg a-tocopheryl acetate/kg for 0, 4, and 9 wk. These fillets were evaluated fresh and after 6 m of frozen storage. Frozen fillets were thawed and stored 3 d at 1 °C prior to analyses. Muscle a-tocopherol of fish fed a vitamin E supplemented diet continuously increased through 9 wk of feeding. Reduced muscle a-tocopherol and moisture, and increased muscle redness and fat were observed in frozen-refrigerated fillets compared to fresh fillets. Thiobarbituric acid-reactive substances were lower in frozen-refrigerated fillets produced from fish fed elevated dietary vitamin E. Proportion of unsaturated fatty acids and omega-3 fatty acids increased as feeding duration increased from 0 to 9 wk.

Supplementing a-tocopheryl acetate (300 and 5000 mg/kg diet) in a trout finishing diet was done to minimize lipid oxidation in oven-cooked fillets and hot-smoked products. Smoking did not affect a-tocopherol content of smoked products compared to raw fillets. Feeding trout diets containing 5000 mg/kg vitamin E increased muscle a-tocopherol content that, in turn, minimized lipid oxidation in oven-cooked fillets produced from fresh and 7-d refrigerated fillets, and in smoked products following refrigerated storage for 8 wk. Dietary vitamin E did not affect fatty acid composition of products from either cooking methods. Oven-cooked fillets produced from 7-d refrigerated, raw fillets and refrigerated, smoked products had lower percents of omega-6 fatty acids and lower omega-3 fatty acids:omega-6 fatty acids ratios compared to fresh, raw samples.

Development of Value-added Food Based on Proteins and Lipids Recovered from Trout Processing By-Products. Based on the initial protein solubility and recovery studies using boneless skinless trout fillets, the trout filleting by-products (frames and heads) were used to recover muscle proteins. A batch system to recover proteins was designed. The recovery system employed a 4.5 L vessel connected with a pH meter and homogenizer for protein solubilization and precipitation steps. The separation was conducted using a batch centrifuge, 6 tubes x 250 ml each tube in one batch. The by-products were homogenized followed by solubilization at pH 2.0, 2.5, 3.0, 12.0, 12.5, or 13.0. Centrifugation at 10,000 x g for 10 min was applied to separate “real by-products” (i.e., bones, skin, fin, scale, insoluble proteins) and fish lipids from the protein solution. The protein solution was recovered and precipitated at pH 5.5 followed by centrifugation at 10,000 x g for 10 min in order to separate the precipitated proteins from the water. Temperature was controlled below 5°C during processing. Ash content in the boneless skinless trout fillets, recovered proteins and the “real by-products” was determined as an indicator of impurity content. The ash content in the recovered proteins and boneless skinless fillets was the same and much lower than in the “real by-products”, indicating good separation of the recovered proteins from the “real by-products”.

The pH of recovered proteins was adjusted to 7.0 and they were used in a silent chopper to develop laboratory protein gels that would allow texture and color evaluation as quality indicators of the recovered proteins. The final moisture content of the gels was adjusted to 80%. The following additives were used during gel formulation: (1) sodium chloride (NaCl) at 2% (w/w), (2) beef plasma protein (BPP) at 1% (w/w) as a protease inhibitor, (3) potato starch (PS) at 3% (w/w) to enhance gel strength, (4) tripolyphosphate (TPP) at 0.3% (w/w) to enhance water retention of the proteins, (5) transglutaminase (T-Gase) at 1% (w/w) to induce protein gelation at low temperature, and (6) sucrose at 4% (w/w) and sorbitol at 4% (w/w) as cryoprotectants to prevent protein denaturation during frozen storage. This formulation resulted in a protein paste that was cooked at 90°C for 15 min. Texture profile analysis (TPA) and torsion test indicated that the gels were firm and cohesive. The tristimulus color values (L*a*b*) indicated that the gels had lower whiteness when compared to the gels made from Alaska pollack grade A surimi. The dynamic test showed that the protein gelation started at 60-65°C, indicating that functional proteins were recovered from the processing by-products.

Protein and Lipid Recovery from Fish Processing By-products: Process Scale-up from Laboratory-batch to Continuous Semi-industrial Application. Based on the initial protein solubility and recovery studies using boneless skinless trout fillets as well as the tests with the processing by-products in the batch system, a continuous semi-industrial system was designed. The system included a continuous homogenizer, two bio-reactors, and two continuous centrifuges. The tests were conducted using trout filleting by-products obtained from our industry collaborator, High Appalachian, LLC (Sophia, WV). Two tests with our industry collaborator, Stephan Machinery (Columbus, OH) were conducted using a continuous pilot scale homogenizer MCH-10. The homogenizer was capable of reducing the particle size to below 0.2 mm and maintaining the flow rate of 120 L/hr, which is suitable for consecutive steps in our process. One test with our industry collaborator, New Brunswick Scientific (Edison, NJ) was conducted using a continuous bio-reactor BioFlo 110 equipped with a 10 L vessel. The bio-reactor was capable of continuous and automatic pH adjustment as well as maintaining the flow rate of 120 L/hr, which is suitable for our process. One test with our industry collaborator, New Brunswick Scientific (Edison, NJ) was conducted using a continuous centrifuge CEPA Z-41. This centrifuge was incapable of separating the protein solution and lipids from the “real by-products” or the precipitated proteins from the water. Therefore, this centrifuge is not suitable for our process. One test with our industry collaborator, Alfa Laval (Richmond, VA) was conducted using a continuous centrifuge GyroTester. This centrifuge was capable of separating our streams. However, the ash content in the recovered proteins was equal to the ash of the trout processing by-products, indicating poor separation. Therefore, more tests have been scheduled for first quarter of 2005 with Alfa Laval using a decanter centrifuge MRNX 438 DD and a clarifying centrifuge. We are currently conducting tests with our industry collaborator, Kendro Laboratory Products (Newton, CT) using a continuous centrifuge CARR Powerfuge Pilot Separation System equipped with vacuum/supernatant pump and vacuum centrate receiver vessel. Another set of separation tests has been scheduled with Westfalia Separator (Northvale, NJ) for the first quarter of 2005.

A second focus of the Aquaculture Food and Marketing Development Project has been to investigate the use of farm raised fish for recreation.

Assess demand and develop marketing strategies for recreational fee fishing packages as complementary recreational activities. Draft of a study which surveyed fishermen who are residents of West Virginia with fishing licenses and fishermen that are not West Virginia residents but have a West Virginia fishing license is nearing completion. The purpose of the study was to assess the level of interest both groups might have in fee-fishing compared to non-fee fishing as a recreational activity. In addition, the study assessed the interest of both respondents in participating in a recreational travel package that included fee fishing, lodging, meals, etc. The results support an interest in both West Virginia fee fishing as a recreational activity and in a recreational fee-fishing package that included lodging, meals, etc. if it were made available.

Data are currently being analyzed for a study that was undertaken to access the level of interest visitors to West Virginia had in recreational fee fishing and a recreational package that included fee-fishing along with lodging, meal, etc. In this study fishing is considered not only as a secondary recreational activity but as a subset or component of a total recreational travel and tourism package for persons interested in vacationing in West Virginia. Preliminary findings show that there is an interest on the part of visitors to West Virginia in a travel and tourism recreational package that includes fee-fishing as one component.

Production of all-female triploid brook trout. Approximately 15,000 all female triploid and diploid brook trout fingerlings were produced at North Carolina State University and transported to the aquaculture facility at Reymann Memorial Farm in May of 2004. In the 11 months since the experiment (two treatments, 4 replicates/treatment) began, there has been no significant difference in growth, survival or feed conversion between the all female triploid and diploid treatments. It is expected that differences among treatments will become most evident during the spawning season in the fall of 2005. These trials will provide a range of baseline production data describing the relative advantages of monosex female and/or triploid brook trout production.

Catch-Related Standards of Quality and Demand Behavior for Fee Fishing Stocking Projects. Hybrid striped bass (HSB) were stocked to compliment the hybrid bluegills that were stocked previously at the same study sites. HSB fishing opportunities were evaluated as part of different recreation program formats (i.e., competitive youth fishing derby, competitive senior’s fishing event, competitive catfish tournament, and drop-in day use). On-site observations and questionnaires were used to evaluate the success of this stocking project at three different WV sites. Among the most desired services reported by anglers include ponds stocked with a variety of fish and at high density levels. HSB were hard hitting and aggressive when first stocked, but later became “hook shy.” The average respondent caught 1 HSB per hour. Over 80 percent of participants were willing to pay $10 per child. HSB were the most likely fish to swallow or partially swallow a hook (16.4% of the time).

Developing and Marketing Fishing Based Travel Packages. This work has only recently begun. The purpose of this study is to develop recreational travel packages that include fee-fishing for visitors who view fishing as a primary motive or as a secondary motive in their decision to visit West Virginia. The research is identifying existing and potential recreational activities, including fee-fishing. The study will also identify lodging and restaurant facilities interested in participating in a travel and tourism recreational package. From the data collected recreational packages for market testing will be developed for specific target markets.

Production of Four Fish Species through Year-Round Operation of a Flowing Water System Utilizing Treated Mine Water.

Four different species have been selected for evaluation in the raceway system utilizing treated mine water at Dogwood Lake. They are: Hybrid striped bass (white bass Morone chrysops x striped bass M. saxatilis) Hybrid bluegill sunfish (Lepomis macrochirus x L. Cyanellus) (male bluegill sunfish x female green sunfish, Largemouth bass (Macropterus salmoides), and Rainbow Trout (Case Western strain). Target harvest density for each raceway unit is approximately 60 kg/m³. Below this density, oxygen concentrations are expected to remain above 70% of saturation and are not expected to impact growth of fish at any point in the system. The number and size of fish in each treatment is described in Table 1. These fish are utilized both for food and recreation and represent the most likely species produced by West Virginia growers for the fishing packages under development. By investigating the growth of these species in treated mine water, opportunities to operate on a year-round basis will be explored and the application of mine waters to support recreational fishing markets will be expanded.

	Hybrid Bluegill (HYBG)	Hybrid Striped Bass (HSB)	Largemouth Bass (LMB)	Rainbow Trout (RBT)
Target Harvest Size (each fish)	1/3 lb	1.5 lb	1 lb	1 lb
Fingerling size (inches)	3to 4	6 to 8	4 to 6	4 to 5
Stocking Number	3000/tank	700/tank	1000/tank	1000/tank

Fish will be purchased from commercial vendors and stocked into the raceway system in September 2006 and grown out for one year. They will be fed a 42% protein, 16 % fat commercially diet by hand with the balance of the ration distributed by pendulum activated demand feeders. The amount fed will be based on the volume of feed distributed to each tank. Feed density (g/L) will be measured and used to convert the volume fed into the weight fed to each raceway unit. Fish will be fed daily to satiation unless water temperatures require less frequent feeding. Fish will not be fed on Sundays or the day prior to sampling. Mortalities will be removed each time the fish are fed and recorded.

Fish will be sampled every six weeks. Total weight will be determined by weighing all fish in each raceway unit. To obtain an average weight, four subsamples will be taken and counted as fish are captured with dip nets and weighed during the process of determining total weight. A subsample of at least 50 fish will be taken to determine condition factor. The length of each fish will be measured to the nearest millimeter, and the average weight of the subsample will be determined. These data will provide direct measurement of feed conversion, average weight, condition factor, and estimation of various parameters during each 6 week period. Fish samples will be taken before stocking, at routine intervals during grow out, and at harvest to assess the potential for bioaccumulation of metals in fish fillets. These assays will be performed according to USEPA guidelines (USEPA 1993).

Production parameters will be compared for each heat tolerant species and with historical data. For instance, historically, for Rainbow Trout reared in treated mine water, the stocking density was 26.4 kg/m³ with an initial loading rate of 0.192 kg/L/min. At harvest, the total net production was 3,275 kg (7,220 lb) with an average loading rate at harvest of 0.40 kg/L/m and a harvest density of 50.2 kg/m³. The survival rate was 98.6%; however, over the duration of the study, 16.6% of the total number of fish stocked were removed for related scientific studies, mortality, and theft. The calculated feed conversion rate (FCR) was 1.4 and the average absolute growth rate over the entire study was 1.52 g/day. The quantitative methods used to calculate productivity-related parameters are:

where M = total fish mass of each segment and V = volume of rearing space of segment.

where M = total fish mass of each segment and Q = water flow rate in each segment.

A schematic of the raceway system is presented in Figure 5. The system consists of two parallel channels on four levels, for a total of eight discrete raceway segments. Each of the eight segments has the following dimensions: 9.1 m (l) x 0.9 m (w) x 1.1 m (d) (30 ft x 3.0 ft x 3.5 ft). The water depth in each segment is controlled by adding or removing dam boards, each of which is approximately 30.5 cm (12 inches) in height. Water is run at a constant depth of 0.9 m (3 ft) at a fixed flow rate of 15.8 L/s (250 gal/min), as determined by rectangular weir measurements. The head loss from the top of the dam boards to the surface of the water in the next segment is 1.1 m (42 inches).

Comparisons of water quality and solids production will be made between heat tolerant species as well as with historic data on rainbow trout collected in previous work. In particular, the following water quality characteristics will be monitored: water and air temperature; water flow rate; five-day biochemical oxygen demand (BOD₅); total suspended solids (TSS); ammonia nitrogen (NH₄⁺-N and NH₃-N); settlable solids; dissolved oxygen (DO); and pH. These parameters are current NPDES regulated water constituents for fish hatcheries in West Virginia. Influent, process water, and effluent turbidity measurements will be taken during field water sampling events. Additionally, alkalinity, acidity, dissolved metals and sulfates will be measured, as each is an important constituent of water chemistry, which can affect fish growth and production. These analyses will add to water quality and fish production data developed over the past three years at the mine water treatment site and will contribute to the modeling project (objective 3). In order to obtain a complete characterization of water quality in the pilot-scale aquaculture system under production conditions, the headbox and final quiescent zones will be outfitted with an in situ water quality monitor capable of measuring pH, temperature, turbidity, conductivity, DO, and depth.

Water samples will be collected at the inlet to the raceway system, the effluent from each quiescent zone and the outlet of each raceway. Emphasis will be placed on sampling of solids from the quiescent zone of the raceways to benchmark differences in solid waste production between individual species. Each parameter will be measured according to the applicable “Standard Method” (APHA 1998) or US EPA (1998) approved analytic method.

The technical challenges of the proposed innovation can be effectively addressed by organizing the research plan into three sequentially major tasks: (1) recycling of FRP scrap for efficient manufacturing of all-recycled and combined recycled and chop strand mat hybrid structural laminates of up to ¼” in thickness, with equal or better properties to laminates made of commercial chop strand mats; (2) manufacturing process development for assembly of core components, consisting of corrugated and straight laminates, and attachment of core to facesheet, to produce prototype sandwich samples with equal or better structural performance than existing designs; and (3) economic assessment of the innovation to demonstrate commercial advantage and market potential. A plan for each of these major tasks with corresponding subtasks is described in this section.

Task 1: Recycling of FRP and Manufacturing and Evaluation of Recycled and Hybrid Laminates: This task will address the following three sub-tasks: (1.1) recycling of FRP materials, (1.2) manufacturing process of laminates, and (1.3) structural evaluations of laminates.

Sub-task 1.1: Production of Recycled FRP Materials - [Performed by KSCI – as Cost-share to this Project]: FRP scrap is the starting point, but it must be processed into materials that are reproducible and which retain fibers long enough to provide the desired physical properties. KSCI will work with Seagull of Volusia County Inc., Edgewater, FL, to produce controlled glass-fibers with statistically defined amounts of either polyester or vinyl ester cured polymers to be used in this study.

Sub-task 1.2: Manufacturing Process of All-recycled and Hybrid Laminates - [Performed by KSCI – as Cost-share to this Project]: The current sinusoidal core configuration and layers of the facesheet for the HFRP sandwich panel are being produced using chop-strand mats (ChSM), for which the WVU team has already established material and system properties. Thus, in this sub-task we will produce flat laminates comparable to ChSM, consisting of all-recycled FRP, if possible, and also recycled FRP and ChSM hybrid combinations. The selected manufacturing process that will be tried is a modified spray-up and contact lay-up combination, based on an existing similar commercial technique (Agarwal and Broutman 1990). This is a low-volume and labor-intense process, but it was selected because of simplicity and manufacturing advantages, including: minimum equipment investment, minimum cost and start-up lead time, low tooling cost, easily implemented by semiskilled labor, flexibility of design, and ability to produce bi-layer laminates (e.g., recycled FRP over ChSM). Since we are interested in reducing material costs, the investigation will include not only glass fibers, but both polyester and vinyl ester resins, suitable for fish tank production.

The feasibility of this manufacturing process will be investigated to produce flat laminates first (Sub-task 1.3), followed by the application of the process to produce and assembled corrugated and flat components of the core, as well as attachment of the core to the facesheet (Sub-task 2.1). KSCI will produce the necessary samples for evaluations by the WVU team.

Sub-task 1.3: Structural Evaluations of Laminates – [Performed by WVU]: The WVU researchers have already obtained material properties for ChSM laminates, which are currently being used for the components of the HFRP panel. This subtask, therefore, will evaluate both stiffness and strength properties of all-recycled and hybrid laminates with three volume percentages of recycled FRP. We describe, materials, testing methods and results.

Materials: The standard core produced by KSCI for fish tanks is made from two 3.0 oz of ChSM which when saturated with polymer cures to a 0.12” cell wall thickness. Similarly, the facesheet consists of three 3.0 oz ChSM layers (0.18”). However, for purposes of material testing, the thickness of the laminate will have to be at least ¼”. Thus, the all-recycled and hybrid laminates will be produced in ¼” thickness, which is equivalent to the volume of a laminate using 12 oz ChSM (about 0.25”). This will allow us to manufacture symmetric hybrid laminates using three volume percentages of recycled FRP, respectively: 25% within two 4.5 oz ChSM, 50% within two 3.0 oz ChSM, and 75% within two 1.5 oz ChSM. It is important to evaluate the best way of manufacturing laminates with recycled FRP, and the material properties that can be achieved; thus, the above four combinations of materials for testing will give us the opportunity to evaluate the concept, and to subsequently select the best process to produce the core and facesheet for the sandwich panel.

Testing and Results: The testing protocol for coupon samples to obtain stiffness and strength will consist of tension, bending, compression, and shear, following modified ASTM guidelines supplemented by proven methods. The number of specimens for each test is estimated as: (6 replications) x (4 sample types) = 24. For tension, samples of 10”x1” will be tested to failure (ASTM D 638-99) to record longitudinal and transverse strains and load-displacement to failure. For bending, samples of 15”x2” will be tested to failure under 3-point loading (ASTM D 790-99), to record mid-span strains and deflections as function of load, following previous work by the researchers (Lopez-Anido, Davalos and Barbero 1995). For compression, the extensive work accomplished and testing tool developed at WVU will be used (Barbero et al. 1999; Makkapati 1994); the samples will be 2”x1”, with strain gages bonded to opposite sides to attain alignment and eliminate bending. Finally, for shear, the Iosipescu test (ASTM D 379-88) method will be used with notched butterfly specimens instrumented with strain gages; the testing tool and the equipment for precise cutting and polishing of specimens are available at WVU. Photographs of ChSM samples tested at WVU are shown in Figs. 2 through 4 for tension, compression and shear, respectively.

Guided by the experiments, the analytical predictions will be based on existing models successfully developed by the researchers. The prediction models for stiffness and strength will be calibrated using the experimental data, which will permit us to make predictions for other possible hybrid laminates to recommend optimum percentages of material combinations, and also to guide the manufacturing of the actual core and attachment to the facesheet.

Task 2: Manufacturing and Evaluation of Prototype Sandwich Samples: This task is organized into 2 sub-tasks: (2.1) manufacturing of core and attachment to facesheet, and (2.2) structural evaluations of prototype sandwich samples.

Sub-task 2.1: Manufacturing Process for Core and Core-to-Facesheet Assembly – [Performed by KSCI as Cost-share to this Project]: Unlike the production of laminates (Sub-task 1.2), the manufacturing of the core is more challenging and will require trials to arrive at a functional solution. The core consists of sequential corrugated and flat components bonded currently by pressure co-curing at contact sections. Both components are produced from ChSM and polymer resin, with a finished thickness of about 0.12”. The core must resist primarily out-of-plane compression and shear, and research at WVU has shown that the thickness of the flat component must be at least about 0.12” (corresponding to 6.0 oz of ChSM) to avoid premature buckling failure. More importantly, the strength of the core-facesheet interface is crucial for product performance; research has shown that a 6.0 oz of ChSM over the inner face of the facesheet can provide an optimum “bonding layer” for embedment of the core into the facesheet by contact-molding. Thus, based on the results of the manufacturing and testing of laminates in Task 1, combined recycled FRP and ChSM hybrids will be manufactured by the spray-up / contact lay-up process (Sub-task 1.2), using the least possible amount of ChSM. The possible manufacturing process for core assembly and core-to-facesheet bonding are described next.

For core assembly, a corrugated component, as thin as possible, will be produced using ChSM (say, 1.5 oz = 0.03”) and existing technology, and while partially cured, recycled FRP will be sprayed over to achieve a prescribed thickness of the hybrid laminate. Similarly, a thin and partially cured flat component will be sprayed with recycled FRP to a given thickness. Then, the smooth ChSM side of the hybrid corrugated component will be co-cured by embedment pressure over the face of the flat containing the recycled FRP. Then, a mirror image of a hybrid corrugated component will be co-cured to the smooth side of the same flat component, with the contact face of the corrugated component containing the recycled FRP (figure 9). This process will be continued to assemble the core for a given product.

For core-to-facesheet assembly, a thin ChSM layer will be applied over the inner face of the facesheet laminate, and then, recycled FRP will be sprayed over to a given thickness, to subsequently embed the core into this “bonding layer.” The rotational and peeling restrain that can be developed at the interface are important to increase core buckling capacity and interface fracture toughness to prevent delamination.

The proposed manufacturing processes will require trials and preliminary evaluations. One concern, for example, is the ability to produce actual-size material (not coupons) of consistent thickness with minimum voids and relatively smooth finished surface; the thickness discrepancies at the contact bonding surfaces between corrugated and flat components can lead to misalignments and dimensional problems, exacerbating core buckling (figure 10). However, the manufacturing of the facesheet using combined recycled FRP and ChSM bi-layer materials should be easier to accomplish.

Sub-task 2.2: Structural Evaluations of Prototype Sandwich Samples – [Performed by WVU]: The WVU researchers have conducted extensive studies of the current product to evaluate its performance to failure, based on experimental and analytical approaches. Based on the most effective manufacturing process defined in Sub-task 2.1, prototype sandwich samples will be produced for testing under compression, flat-wise tension, and bending, with materials and testing methods and results as described next.

Materials: The specimens for compression and flat-wise tension will consist of single-cell units, cut from a sandwich panel. This in-plane symmetric unit of 4”x4” (see Fig. 6) is considered a “representative volume element.” For bending, the sample will consist of a 4” wide strip extending 28” along the sine-wave of the corrugated component, resulting in seven contiguous unit cells. The thickness of all the samples will be 2”, in order to correlate results with existing information obtained for similar samples, and the lay-up of the facesheet will consist of combined ChSM and attached inner “bonding-layer” containing the recycled FRP.

Testing and Results: The testing protocol for strength evaluations will consist of compression, flat-wise tension, and bending, following modified ASTM guidelines and previous methods developed by the WVU team. The number of specimens for each test is estimated as: (6 replications) x (2 sample types, possibly) = 12. The bending failure load is sensitive to the interface bonding layer effect and the contact thickness of the core components, which maybe variables that will need to be valuated experimentally, and predicted by an existing failure criteria using FE modeling.

Task 3: Economic Assessment and Commercial Potential: This third and final task of the Research Plan will address economic aspects for product commercialization. The aquaculture industry will benefit from the present proposed study to substantially reduce costs, while promoting recycling technology of discarded FRP.

Based on the current KSCI core geometry, we anticipate that the core wall thickness, containing about 50% recycled FRP and polymer resin by volume, will have to increase by some percentage to compensate for the expected lower compression and shear strengths of the hybrid laminate. Thus, assuming all currently used materials are priced equally, and further assuming that the value of recycled FRP will be $0.20/lb or $400/ton including transportation and processing, the price of the hybrid laminate, in relation to a currently used 4.5 oz of ChSM priced at $1.19/board-foot, would be about $0.46/bf, representing a substantial savings of $0.73/bf or 61%.

Task 1 – Continuing Development of Software Tool

It is proposed that the software tool continue to be developed in an Excelä programming environment using the VBA programming language. This software platform offers an extensive range of built in functions and user-interface tools and produces a user-friendly interface.

The next phase of the modeling is to expand the analysis to multi-tank raceways. The approach will be to develop software that gives the user as much flexibility as possible in configuring the raceway. The logic flow diagram for the proposed software is illustrated in Figure 11. This figure shows how a user may specify any raceway system consisting of any number of tanks in series with multiple parallel raceways. The user will also provide information pertaining to the location of the raceway, e.g., elevation, seasonal temperatures, water quality, etc. It is envisioned that the software will give the user the capability to simulate scenarios in which fish cohorts may be placed in different tanks at different times. Therefore, the user may input information at any time during a scenario regarding the movement of fish from one tank to another, and the partial or total removal of fish from a tank. The program will then simulate the growth, optimal feed rate, oxygen consumption, nitrogen production, oxygen replenishment via weirs and/or other reoxygenation technologies, and economic parameters such as the cost of feed and fingerlings and the revenue from fish sales.

In order to allow the user to obtain growing scenarios that are as realistic as possible, the software will be written as an event-based tool. Therefore, the user (fish grower) will be able to enter information regarding fish movements within the raceway at any time during the simulation. As much flexibility as possible will be built into the software to enable the user to go back and change the time at which a fish moving event (purchase of new cohort, movement of cohort from one tank to another, or the removal and sale of a whole or part of a cohort) occurs. Optimal feeding rates will be determined based on water temperature. The growth of the different cohorts will be predicted by the software, and basic economic information on the cost of feed, cost of fingerlings, and revenue from sales as a function of time will be included in the output.

Figure 11: Schematic diagram of information flow in the multi-tank raceway simulation software

Task 2 – Analysis, interpretation, and synthesis of data from on-going research

In developing the single-tank model, in precious work (FY 2004), certain gaps in information regarding the growth of trout have become apparent. For example, Soderberg (1995) discusses the growth of a variety of fish in terms of the water temperature and the corresponding feeding rate required to obtain optimal growth. This information forms the basis of growth prediction in the software. Hardy and Barrows (2001) indicate that growth may be tailored by changing the feed rate from the optimal level. In order to implement this additional level of sophistication in the software, a mathematical model must be developed to capture the physics/biology of the process. The basis of such models for salmonids may exist or may be available through the correlation and interpretation of data in the literature or generated by researchers at WVU. Similarly, the way in which the distribution of fish sizes changes during growth is another area in which anecdotal information is available but no suitable model exists. A substantial body of data exists for both Wardensville and Dogwood Lake raceways, however, the reduction and analysis of this data to provide a suitable model for the software has not been attempted. The purpose of Task 2 is to provide support in correlating and interpreting data, relevant to the growing of trout, generated from various researchers and activities at WVU. In addition, models will be developed based on these data and/or other published data and these will be implemented in the software.

A polyhouse (10’ x 20’) will be constructed to house 4 sets of 3 channels each. Experiments will contrast nutrient removal and growth of warm and cool season plants in the polyhouse, in the settling pond adjacent to the building as well as in the raceway building. Culture conditions that optimize nutrient removal and biomass accrual will be selected based on the experiments currently underway.

Each experiment will consist of 24 channels, 12 in the raceway building and 12 in the polyhouse. Each plant species (watercress, lettuce, basil and dill) will be placed in 3 channels in each location. Effluent from the raceways will be pumped to the experimental channels in each facility with the optimal water velocity determined from our current experiments. Additionally, this experiment will be repeated in the settling pond which receives aquaculture effluent. The same plant species, in the same culture medium, at the same density will be used. A schematic of the experimental layout is given in Figure 12.

The entire experiment will be repeated in each of the four seasons to quantify the unique combinations of daylength, light intensity and air temperature found in each season. Air temperature, water temperature and light intensity will be monitored in the existing building, in the polyhouse and at the settling pond edge. The following water quality characteristics will be monitored: water temperature, water flow rate, ammonia, nitrate, nitrite, total phosphate, and pH. Samples will be collected at the end of the raceway system and then at the bottom of each of the experimental channels. Water samples will be taken at roughly 3 week intervals. Biomass and nutrient concentrations (total C, total N and total P) of the plant material will be determined at the beginning and end of each experiment to determine growth and nutrient sequestration. Each parameter will be measured according to the applicable “Standard Method” (APHA 1998) or US EPA (1998) approved analytic method.

Figure 12. Schematic of existing raceway building and channels, along with polyhouse and settling pond.

The overall objective is to scale up the protein and lipid recovery batch process to continuous semi-industrial application. Specific objectives are: (1) equipment purchasing, installation, training, and set-up; (2) optimization of by-product/water ratio; (3) optimization of pH, agitation, homogenization and viscosity; (4) optimization of reaction time and flow rate (residence time); (5) determination of protein and lipid recovery yields; (6) optimization of continuous separation of proteins, lipids, water, and insolubles; (7) texture and color evaluation of gels developed from recovered proteins.

A two-dimensional (2-D) process scale-up is proposed, from laboratory-scale batch to semi-industrial continuous application. The 2-D means that the scale-up will involve simultaneous increase of capacity, from 1 lb/day to 26 lbs/hr and change of the operation mode, from batch operation to continuous operation. The WVU Research Corporation has filed a patent application on the investigator’s behalf to the U.S. Patent Office. The patent application is based on this system.

The investigator has already designed a system that will be capable of accomplishing the proposed scale-up. The continuous system will be based on a by-product homogenizer (step 1), two bio-reactors (steps 2 and 4), and two continuous centrifuges (steps 3 and 5).

Most of the necessary equipment has been separately tested with our industry collaborators. However, the equipment have not been tested when separate machines were connected to each other. A by-product homogenizer by Stephan Machinery (Columbus, OH) has been tested. Bio-reactors by New Brunswick Scientific (New Edison, NJ) and Sartorius BBI Systems (Allentown, PA) have been tested. Continuous centrifuges by Alfa Laval (Richmond, VA), New Brunswick Scientific (New Edison, NJ), and Carr Centritech Separation Systems (Newtown, CT) have been tested.

The funds from FY 2004 will be used to purchase by-product homogenizer and the bio-reactors. The funds requested in this proposal (FY 2005) will be used to test some of the equipment when connected together and purchase and set up the continuous centrifuges.

(1) Equipment will be purchased, installed and set up in the Meats Laboratory in the Division of Animal and Veterinary Sciences. The analyses will be conducted in the investigator’s food science and technology laboratory in the Division of Animal and Veterinary Sciences. The investigator has sufficient laboratory space to accommodate the equipment and has the necessary instruments to test the recovered proteins and lipids.

(2) In our experiments we have learned that during the pH shifts (steps 2 and 4) the viscosity increases significantly at two pH ranges 3.5-4.5 and 9.5-10.5. Viscosity is critical for efficient separation by centrifugation (steps 3 and 5). Therefore, it is necessary to add water to the by-products during homogenization (step 1). We determined that for batch operation at the lab scale best by-product/water ratio was 1/9. The by-product/water ratio will be tested for the continuous system in order to optimize this factor for best protein and lipid recovery. The water added to the proposed system will be recovered in second centrifugation (step 5). It was determined in our experiments that the recovered water is protein-free and clear, and therefore, can be recycled in the system.

(3) The pH is critical for protein solubility and precipitation (figure 14), and thus, directly relates to the recovery yield (figure 13). Therefore, various pH’s will be tested. Homogenization reduces particle size, and therefore, facilitates reaction between proteins and lipids with water. In the continuous system, the homogenized slurry (water + by-products; step 1) will be pumped to bio-reactors. Therefore, homogenization will be critical for efficient protein solubility and lipid separation as well as prevention of tube clogging during pumping. Bioreactors are equipped with agitation, which facilitates reaction between proteins and lipids with water. At the same time, agitation is affected by the slurry viscosity. Therefore, the agitation and viscosity will be optimized for efficient reaction between proteins and lipids with water, but without promoting excessive foaming.

(4) Objectives 1-3 are related to the flow rate, which is defined as the volume of the solution that will flow through the recovery system (two bio-reactors + two centrifuges). In other words, how long the solution needs to stay in the bio-reactor (residence time) for the reaction between the proteins and lipids with water to occur. The shorter the residence time, the more efficient the continuous system becomes. In other words, more solution per hour can be processed by the system. Therefore, following completion of objectives 1-3, the flow rate will be optimized for the shortest residence time (highest flow rate) that will result in highest protein and lipid recovery. From our experiments with the lab-scale batch operation, we have determined that 10 minutes is sufficient for the reaction between proteins and lipids with water to take place. Therefore, the recovery of 26 lbs/hr mentioned above is based on the 10-min reaction time.

(5) Protein and lipid recovery yields will be determined as in previous experiments with the lab-scale batch process (figure 13).

(6) The centrifugation time and g force will be tested for efficient continuous separation of proteins, lipids, insolubles and water. This objective is related to the objective (2) and (3).

(7) Protein gelation is the most critical quality parameter for development of restructured value-added food products. Protein denaturation destroys gelation ability. Proteins undergo ir-reversible denaturation when subjected to heat or ionizing radiation, and reversible denaturation when subjected to extreme pH. Therefore, gelation properties of recovered muscle proteins will be evaluated using Bohlin dynamic rheometer. Texture properties of the samples developed from recovered proteins will be evaluated using Hamann gelometer, Kramer shear, and texture profile analysis (TPA). The color properties will be evaluated using L*a*b* Chroma meter.

The need for technology transfer activities remains strong. Results from a wide series of projects must be presented to existing and prospective producers, and the state agencies influencing aquaculture development. Presentations are also made to a diversity of groups at state and national meetings. Requests for literature and information have doubled since the inception of the project. There is greater demand for site visits to assess opportunities, and assist individuals growing or utilizing farm raised fish. Administrative duties continue to require the attention of the Extension Specialist in his role as Principal Investigator making it difficult to meet all requests and reach out to new stakeholder groups.

Consistent with this demand and the selected areas of emphasis, two technology transfer positions have been established. Dan Miller, a Research Associate within the Division of Resource Economics is delegated tasks relating to development of the mine water resource. Rodney Kiser, a Research Assistant within WV Extension Service is delegated tasks relating to the use of farm raised fish in recreation. Each position covers an area distant from Morgantown and complements the efforts of the Extension Specialist – Aquaculture.

Development of the Mine Water Resource. Mine water has emerged as the most important source of water for commercial production of salmonids in West Virginia. Each site must be evaluated on its unique topography, water quality, water volume, etc. We continue to engage two large coal companies and numerous mine sites. We will continue to work with coal companies and economic development agencies the step by step process of site assessment and education required for development of the mine water resource. We will also coordinate with processing plants and others to assist with determination production capacity and appropriate facility design once a site has been chosen for development. The individual responsible for this area of work will coordinate with various investigators to develop workshops, a newsletter, and publications useful for development of the mine water resource.

Aquaculture and Recreation. Most fish farmers in West Virginia sell their fish in the recreational market. Approximately 300,000 lb is sold to fee fishing businesses around the state. In addition, live fish are sold to fishing clubs, large companies, housing associations, and private individuals for recreational use in both private and public waters. Specific efforts have recently developed creating an increased demand for work in this area. Development of fishing packages centering at lodging facilities at Pipestem and Stonewall Jackson Lake Resort has begun. We are collaborating with Senator Walt Helmick regarding a fish stocking initiative to enhance tourism. Building upon the data developed in previous research, it is timely that this information be combined with the experiences of fish farmers serving the recreational industry and extend it to a wide variety of recreational stakeholders. Such stakeholders would include resorts, attractions, fishing clubs, communities developing strategies to enhance tourism, and the general public. Site visits and assistance to farmers targeting the recreational market will also be an important component of this work. There is opportunity to develop partnerships between segments of the tourism industry, fish farmers, fisheries managers, and the resource base to respond to the recreational opportunities described by marketing research. The aquaculture facility at Reymann Memorial Farm will increasingly be utilized as an educational venue.

Implementation of technology transfer activities is to disseminate information generated by this project to the aquaculture industry in Appalachia, to government agencies with aquaculture-related responsibilities, and to the general public. The investigators will continue to collaborate with the Freshwater Institute’s Aquaculture Program, the West Virginia Department of Agriculture, the West Virginia Aquaculture Association, and other organizations to deliver research findings in the most user friendly manner possible. In January we will host the Aquaculture Forum – an annual meeting designed to reach aquaculture stakeholders and present a program of research results and practical commercial perspectives.

Production of Four Fish Species through Year-Round Operation of a Flowing Water System Utilizing Treated Mine Water.

Rainbow trout grow well in treated mine water at Dogwood Lake on a seasonal basis. Unfortunately water temperature may exceed 75 F during the summer months, resulting in temperatures unsuitable for production of most trout. As a result, fish must be removed from the system during the summer months. The ability to grow or maintain fish populations year round are central to obtaining maximum production and marketing opportunity from the system. Production of other fish species or strains tolerant of warm summer water temperatures is proposed in this task. The following species have been selected for study.

Hybrid striped bass (white bass Morone chrysops x striped bass M. saxatilis) are typically grown in ponds throughout the southern United States. In the Northeast US, they are grown in cages, recirculating systems, and flowing water systems. Hybrid striped bass are popular both as a sport fish and as a food fish. They readily adapt to pelleted feeds, convert well, and grow rapidly. Retail prices for hybrid striped bass are usually double or triple the price of rainbow trout. Demand for hybrid striped bass as a food fish has been increasing. Hybrid striped bass is a good candidate for investigation into economic maximization of treated mine waters, particularly those with variable temperatures, due to the hybrid striped bass’ wide tolerance to a range of temperatures and salinities.

Hybrid bluegill sunfish (Lepomis macrochirus x L. Cyanellus) (male bluegill sunfish x female green sunfish We expect to improve both feed conversion and survival by growing hybrid bluegill in flowing water systems. Little information is available for production of hybrid bluegill in flowing water systems. These fish are especially well suited for recreational fishing for families with children as they aggressively bite even the unbaited hook. They readily adapt to pelleted feeds and demand a retail price/lb similar to hybrid striped bass. Fingerlings are easily produced by WV farmers.

Largemouth bass (Macropterus salmoides) are a popular fish in recreational markets. They do not normally take a pelleted feed unless trained to do so. Once the fingerlings are trained to feed on a pellet they grow well to a 1 lb size and command prices comparable to hybrid bluegill and hybrid striped bass. Ethnic asian markets represent a potential food fish market. Like hybrid bluegill, little information is available on the production of largemouth bass in flowing water systems.

Rainbow Trout (Case Western strain)– A vendor in Pennsylvania has maintained the Case Western strain of rainbow trout (Oncorhynchus mykiss) developed at Case Western Reserve University about 20 years ago. This unique strain was selected to survive summer temperatures in ponds at this latitude and is listed in the National Fish Strain Registry. This strain is reputed to withstand summer water temperatures over 70ºF. It may grow more slowly and is less uniform in size than the Kamloops strain used in previous studies at Dogwood Lake. Data regarding production of these fish under the range of temperatures expected in this experiment has not been obtained. If they are tolerant of warm temperatures, the growth rate and feed conversion will be higher than expected based on growth predicted by the software model currently under development.

Outcomes of this study will provide continuity in water quality and fish production data in mine water and support economic demand and marketability studies to ultimately develop recommendations for the further utilization and commercialization of mine water aquaculture. For instance, data developed in this study can be used to determine if it is more profitable for mine companies, or others utilizing treated mine water, to grow fish for the recreation markets (i.e., stocking streams, pay lakes, and recreation lakes) rather than as food fish.

The numerous favorable attributes of HFRP sandwich panels make them ideally suitable for aquaculture raceway systems and other similar applications in aquaculture industry. But the most significant barrier to the widespread adoption of HFRP systems is probably cost. Both previous work (FY 2003) and the present proposed effort (FY 2005) are directed to reducing costs. Work described in this proposal will incorporate recycled materials in significant amounts (about 50%) in the manufacturing of HFRP sandwich panels. The manufacturing flexibility of the KSCI panel, whereby the top and bottom facesheets and the core can be produced separately, allows for versatility of incorporating recycled materials for each component by distinct processes.

Recycled Materials: We propose to use industrial scrap of FRP materials that are currently simply being damped in land fields. The abundant availability of these materials combined with recent technologies and the strong support of two industries involved in this initiative make this proposal timely and of great potential for success.

The FRP manufacturing scrap materials produced by the composites industry in the US in 2003 is estimated to be 50 million pounds. Until recently, there were no effective methods to recycle FRP thermoset materials, but at present, Seagull of Volusia County Inc., a Florida company collaborating in this project and partnering with KSCI, has developed a shredding technology to produce granulated FRP materials. The shredded material contains fibers with some amounts of attached cured polymers, and based on favorable results obtained by KSCI over the last 2 years, the recycled material has shown to be consistent in particle size and properties, with promising results similar to those of commercial chop-strand mat. KSCI has stock piled several tons of material for ready use in this project.

Hybrid HFRP Materials: Our proposal is to replace approximately 50% of the Chop-Strand Mat (ChSM) currently being used in HFRP construction with recycled FRP. The technical efforts will involve: (1) implementing a manufacturing process by a modified spray-up/contact lay-up technique; (2) producing hybrid laminates with equivalent properties to commercial ChSM; (3) developing a manufacturing process for assembly of core components and core-to-facesheet bonding for HFRP panel production; (4) evaluating coupon and component-level specimens to demonstrate performance in relation to existing data; and (5) conducting an economic analysis to demonstrate the economic advantage of the innovation and the commercial feasibility of the product.

Full-scale Tank: The new hybrid material system developed with FY 2005 funds will be combined with the connector system developed under previously manufacture a full-size HFRP tank, which will exhibit both efficient assembly using flat panels and mechanical connectors, and FRP recycled materials. This tank will probably be installed at Wardensville or other site for field evaluation.

Significance and Future Potential. Considering the above cost estimate for the sandwich product, the current cost of a commonly used sandwich panel for fish tanks will be reduced by about 50%. Neglecting labor costs differentials for now, this large materials cost reduction is significant and probably sufficient to reach a fully competitive price target with concrete. But together with materials cost reductions and mechanized production of the product, yet to be undertaken, KSCI will have the potential of actually manufacturing the first truly price-competitive FRP raceway system. This project will provide the opportunity to develop a commercially competitive raceway system for the aquaculture industry, leading to a truly engineered product of modular construction and long service-life. Thus, success in this project will not only repay the USDA investment, but also promote an industry of great benefits to aquaculture, while developing sustainable technologies and economic prosperity nationwide and particularly in the Appalachian region.

The development of this software is an important activity that will provide a useful and practical tool for estimating the profitability of existing and proposed raceway systems. The software can be used to help entrepreneurs and investors make predictions about proposed raceway systems by evaluating the economics of typical operating scenarios and quantify the revenues and costs associated with the operation of the raceway. In a similar manner, the software will allow current fish growers to evaluate different stocking, feeding, and harvesting regimens to identify the optimal operation of existing raceways. Moreover, the sensitivity of results to changes in parameters including feed conversion rates, stocking rates, and oxygen supplementation may also be examined.

The purpose of Task 2 is to analyze, interpret, and synthesize data from on-going and/or new research projects that relate to fish growth. This work is essential to ensure that models of fish growth take into account the effects of sub-optimal feeding and also, if possible, account for the spread of the distribution of fish sizes within a given cohort. This work ties in with the on going research at the Wardensville and Dogwood Lake facilities conducted by the Extension team.

There is increasing concern regarding nutrient loading from various sources including aquaculture operations on receiving streams. Both Selong and Helfrich (1998) and Loch et al. (1996) demonstrated deleterious effects of flow-through aquaculture systems on downstream benthic communities. In addition to local impacts, nutrient inputs from upstream sources have negative effects on downstream estuaries. In the Chesapeake Bay, as well as other estuaries, there are strong linkages between increases in nutrient loading and large algal blooms that lead to anoxia and toxic or harmful impacts on fisheries, human health and recreation (Anderson et al. 2002). As a result, the states with major rivers flowing into the Chesapeake Bay have pledged to reduce nutrient loading to the bay. Aquaponics, the simultaneous culture of fish and plants has the potential to reduce nutrient export from fish production as well as provide an additional income source through the sale of plant products.

Filleting trout requires removal of bones, skin, head, and viscera (by-products). Mechanical filleting of 100 lbs of trout yields approximately 40 lbs of fillets and 60 lbs of by-products. The 60 lbs of by-products contain approximately 20 lbs of meat and 5 lbs of fish oils (lipids). The by-products are reduced to animal feed or are land-filled. Fish processors incur expenditures to remove processing by-products from their plants. The by-products are also an environmental bio-burden.

The development of a technology that allows recovery of proteins and lipids from by-products will increase profitability of fish processors and broaden their product offerings as well as may create new jobs in West Virginia. The use of recovered proteins and lipids for human food instead of for animal feed or land-filling will increase the value of these materials. Therefore, human food products developed using proteins and lipids recovered from processing by-products are a typical example of value-added food products.

This research will allow implementation of the protein and lipid recovery technology developed in our lab to semi-industrial application. If this research proves that this technology can be implemented at the semi-industrial scale, then the next step would be full-scale industrial production. Therefore, the proposed research will fill the gap between technology development at the university laboratory bench-top scale that our laboratory has done so far and full industrial production that is still a far perspective.

The proposed research will enable the investigator to establish a protein and lipid recovery system that to the investigator’s knowledge has not been previously constructed. Therefore, our laboratory would have a privilege of being at the forefront in protein and lipid recovery research. This would result in increased feasibility for attracting external research funds from federal sources such as USDA-NRI (program 71.1 – value-added product research: food characterization/process/product), Northeastern Regional Aquaculture Center (NRAC) and National Science Foundation (NSF). The USDA-NRI and NSF promote young investigators. Therefore, the investigator would qualify for the New Investigator Award from both agencies due to investigator’s Assistant Professor status.

The proposed recovery system will be universal, meaning that the proposed system will be as applicable to any other food animal species as to trout, as long as the isolectric point and solubility of the other species’ proteins are determined. Therefore, this will open additional external funding opportunities to agencies such as U.S. Egg and Poultry, Fats and Proteins, and National Fisheries Institute. Since the proposed system will be at the semi-industrial scale, the investigator anticipates increased interest and funding from private food animal processing businesses. Since the proposed system will utilize food animal processing by-products that otherwise would be disposed of being an environmental bio-burden, funding from the EPA may be feasible.

Production of Four Fish Species through Year-Round Operation of a Flowing Water System Utilizing Treated Mine Water.

In previous proof-of-concept work conducted by Viadero and Tierney (2003), it was shown that the use of treated coal mine water for rainbow trout (Oncorhynchus mykiss) culture in a cage was technically feasible, though only a fifty fish bioassay was grown and no work on production related issues was conducted. To further advance the use of treated mine water, an under-utilized water resource throughout Mid-Appalachia, work was conducted to assess the effects of using treated coal mine water for the intensive production of rainbow trout in a flow through system (Viadero and Tierney, 2004). During this study, comprehensive water quality data were collected to supplement fish weight and length data taken during routine monthly sampling events. The eight thousand fish grew well in the raceway system over the nine months of production, where a feed conversion ratio of 1.4 and a condition factor of 5.1x10^-4 were measured with stocking and harvest densities of 26.4 and 50.2 kg/m³, respectively. Further, total net production was 3,275 kg (7,220 lb) with 98.6% survival. Throughout the study, dissolved ion concentrations (Fe, Al, Mg, Ca, and SO₄) often exceeded recommended limits. Further, elevated ammonia nitrogen concentrations generated from a component of the mine water treatment process were identified as a potential limiting factor for aquaculture development. However, when the non-ideal effects of high ionic strength and the speciation of dissolved metal–ligand complexes were taken into account, the concentrations of free metal ions were within recommended limits.

Though the use of treated mine water for the culture of rainbow trout has been investigated in both published and unpublished studies, there has been very little investigation into the use of treated mine water for warm-water fish species. Hybrid bluegill sunfish grown in ponds in WV had a feed conversion of about 4.0 and a survival rate of about 55% (Semmens 2005). These values are low and may be improved in a flowing water system.

Quantative description of water quality and production relationships have been described by Soderberg (1995), Klontz (1991), and Piper (1982). The information has not been integrated into a tool easily utilized by growers and educators and made readily available to the public.

Aquaponics has been developed in conjunction with recirculating aquaculture systems (RAS). Rakocy et al. (2000) developed a system that produced 3.1 mt of tilapia and 1,248 cases of lettuce over a 2.5 year period. Adler et al. (1996, 2000) developed an off-line system that uses trout effluent to grow lettuce and basil. In this system NO₃ concentrations were reduced from 25 to 3 mg·l^-1 and PO₄ concentrations were reduced from 0.7 to <0.001 mg·l^-1. RAS are characterized by high nutrient concentrations and low volumes when compared with flow-through aquaculture systems.

The pH-driven protein recovery. Protein solubility is lowest at a protein’s isoelectric point (pI) (Srinivasan 1996). The pI is the pH at which the protein molecule’s net electric charge is zero. As pH diverges from the pI, the increased protein-protein electrostatic repulsion facilitates protein-water interaction, resulting in protein solubilization (Srinivasan 1996). Conversely, as pH approaches pI, the decreased protein-protein electrostatic repulsion decreases protein-water interaction, allowing for protein-protein hydrophobic interactions, thereby leading to protein precipitation (Srinivasan 1996). Precipitated proteins can be separated from lipids, debris (bones, skin, etc.), and water by centrifugation. This isoelectric solubilization-precipitation cycle can be used to isolate proteins and lipids from the trout processing by-products.

Gel-forming ability. To be useful in making surimi seafood products, muscle proteins recovered from the by-products must retain their gel-forming ability (Lanier 2000). Heat-induced gelation is a result of protein denaturation, leading to inter- and intramolecular covalent and non-covalent interactions (Lee and Lanier 1995). Solubilized proteins undergo denaturation followed by an ordered aggregation to form a gel network (Srinivasan 1996). Therefore, solubility and denaturation are the critical prerequisites for heat-induced protein gelation.

Fish oil and omega-3 fatty acids. Fish oil contains high concentrations of omega-3 polyunsaturated fatty acids (w-3 PUFA). In 2002, the Food and Drug Administration (FDA) approved a health claim for the w-3 PUFA. According to the FDA, “consumption of w-3 PUFA reduces the risk of coronary heart disease”. This approval created a niche market in the functional foods and dietary supplements arenas for fish oils. The concentrations of the w-3 PUFA in a fatty acid profile (FAP) of fish oil vary depending on species, diet and processing. Therefore, determination of the FAP is critical.

Current research in the field. Hultin and Kelleher (1999) applied the isoelectric solubilization/precipitation to recover muscle proteins from mackerel and demonstrated that solubilization of mackerel muscle proteins at pH 2-3, followed by precipitation at pH 5.5 and centrifugation, resulted in high recovery of muscle proteins and lipids. Choi and Park (2000) applied the isoelectric solubilization/precipitation to recover muscle proteins from Pacific whiting (similar pH as Hultin and Kelleher), which increased muscle protein recovery by 80% as compared to conventional surimi production (sequential washings with water). However, the poor texture of the surimi seafood product prepared from Pacific whiting proteins recovered at acidic pH was attributed to activation of proteases at acidic pH. Yongsawatdigul and Park (2001) further investigated protein recovery of fish muscle using an alkaline pH, which resulted in improved texture of the surimi seafood product.

There are no reports on application of the isoelectric solubiliation/precipitation to recover muscle proteins from food animals processing by-products, including trout. To the investigator’s knowledge, a construction of the continuous system at the semi-industrial scale using isoelectric solubilization/precipitation of muscle proteins has not occurred.

Production of Four Fish Species through Year-Round Operation of a Flowing Water System Utilizing Treated Mine Water.

An experiment to evaluate two commercial diets for production of rainbow in treated mine water is currently underway. One diet treatment contains fish meal and the other does not. The evaluation will include estimates of feed conversion ratio, growth rate, cost, quantity and characteristics of solid and dissolved waste production, and the concentration of organochlorine compounds (e.g., polychlorinated biphenyls, dioxins, etc.) in fish flesh grown in WVU’s mine water raceway

One highlight of the aquaculture research at WVU has been the development of raceway systems from Honeycomb Fiber-Reinforced Polymer (HFRP) sandwich panels. The three successful projects at Dogwood Lakes, Wardensville (Reymann Memorial Farm), and Warwick Mine have shown the efficiency and versatility of HFRP construction for raceway systems to be installed either resting on ground or as in-pond floating systems. Initially, two systems were developed using built-in construction of complete tanks manufactured at the factory and shipped to the site for field installation (Dogwood Lakes and Wardensville). Then later, the floating system at Warwick Mine was field-assembled from flat panels using mechanical connectors to join the pre-manufactured component panels. The objective was to explore a simplified manufacturing process to reduce costs; this concept was shown to be successful and served to focus the research work with the development of modular systems assembled from flat panels. This work has concentrated on numerical simulations of efficient connector methods to chose and test a “universal connector” for product assembly by the producer, using simple tools and easy-to-follow instructions for unskilled labor. Tests of prototype connected samples have shown the potential efficiency of the proposed universal connector to attach vertical-to-horizontal panels.

Successful and progressive results have shown convincingly that the HFRP technology offers potential for aquaculture development and commercialization in West Virginia and other places (Vantaram et al. 2002). However, one disadvantage of using HFRP is the relatively higher cost of the product in relation to similar designs made of reinforced concrete. Although the advantages of HFRP systems far surpass the limitations of concrete, including life-cycle costs, most producers make decisions on first-cost basis, and therefore, there is a need to reduce HFRP costs to make it competitive with concrete. It is interesting to note, however, that the costs of currently available pre-manufactured aquaculture tanks, using for example fiberglass construction, are in most instances comparable to present costs of production of HFRP systems, but unlike existing products, the quality and performance of the HFRP designs are much superior to commercial systems.

Over the past 15 months, an Excelä-based software tool was developed that predicts the performance of a single tank in a raceway system. The software is currently being tested and reviewed both within and outside of WVU. The approach has been to develop a user-friendly, event-based tool that allows the user great flexibility in entering data and extracting results. The software estimates optimal growth rate, optimal feed rate, oxygen requirements, and waste nitrogen production in a single tank. Required input data include, seasonal temperature, composition of feed, number and length/weight of initial fish population, etc. The user enters data and simulates the desired results by moving through the 6 buttons at the top left hand corner of the main input form. Drop down menus allow input of data. Results are displayed on the main form as they are calculated. The results and input data are presented in the form of monthly reports that detail fish growth, feed requirements, oxygen usage (Soderberg 1995, Klontz 1991), nitrogen generation, and a variety of other information. All the data shown on the results form is also automatically downloaded to an excel spreadsheet for easy access and post processing by the user, if required.

Current work will quantify culture variables necessary to optimize nutrient uptake and watercress production using aquaculture effluent from a flow-through facility as the nutrient source. The installation of the 30 experimental channel array is 70% complete. The system has been designed, all of the channels have been constructed and the plumbing will be completed shortly. Initial germination trials of the watercress seed have been conducted to determine percent germination and seed viability. Construction of the rafts which will be used as the substrate to grow the plants on are near completion.

Up-to-day developments have resulted in a submission of a patent to the U.S. Patent Office by the WVU Research Corporation on the investigator’s behalf.

We have developed a batch operation for protein and lipid recovery at the laboratory scale that allows us efficient recovery of functional muscle proteins and lipids from trout. The batch operation unlike continuous operation mode is defined as a cyclic operation that requires repetitive cycles of (1) loading substrate (fish processing by-products), (2) processing (isoelectric solubilization and precipitation of fish muscle proteins), and (3) unloading the products (recovered fish muscle proteins and lipids). In contrast to batch operation, continuous operation mode allows continuous feeding of substrate, continuous processing, and continuous harvest of the products. Therefore, the continuous operation mode is a preferred operation type for the protein and lipid recovery from fish processing by-products. The protein recovery yield with our technology is approximately 90% on dry weight basis (figure 13). The recovery technology is based on isoelectric solubilization and precipitation of trout muscle proteins (figure 14). The recovered trout muscle proteins retain their functionality – gelation, which is critical in development of restructured value-added food products. We have also developed protein gels from recovered proteins. The laboratory-developed gels mimic restructured value-added foods and allow scientific determination of texture and color properties, which are the two most important quality attributes for these foods. The texture of our gels developed from trout proteins exhibited superior properties in comparison to gels developed from highest grade commercial Alaska Pollack surimi. Color properties of both gels were comparable.

Based on our experiments, we developed the following five steps (figure 15) necessary to recover muscle protein and lipids: (1) homogenization that simplifies sample handling and increases surface area of proteins and lipids, and therefore, facilitates interaction between proteins and lipids with water; (2) first pH shift that results in protein solubilization due to increased electrostatic4 interaction between proteins and water, and facilitates separation of lipids from water due to increased hydrophobic interaction between lipids; (3) separation by first centrifugation that results in bottom fraction (“real by-products” – bones, skin, and insoluble proteins), middle fraction (muscle proteins solubilized in water), and top fraction (fish lipids rich in omega-3 fatty acids as confirmed by our experiments), (4) the middle fraction is recovered is subjected to the second pH shift that results in isoelectric precipitation of muscle proteins due to increased repulsion between protein and water and increased hydrophobic interaction between proteins, and (5) separation by second centrifugation that results in separation of precipitated functional muscle proteins from water. The water separated in this step is protein-free and clear, and therefore, can be recycled in the process.

The physical facilities of West Virginia University will be utilized for developing, analyzing and reporting the study. Facilities of the Davis College of Agriculture, Forestry and Consumer Sciences will draw upon resources from the two divisions of Animal and Veterinary Science, and Plant and Soil Sciences. The Department of Civil and Environmental Engineering, in the College of Engineering, and the West Virginia Extension Service will provide additional resources. Personal computers and software provided by the institution will be used in implementing this project.

The protein recovery experiments will be performed in the Division of Animal and Veterinary Sciences (A&VS). Dr. Jaczynski has a sufficient laboratory space in the A&VS, which houses the following equipment spectrophotometer, SDS-PAGE and isoelectric focusing cell, homogenizer, ultra-speed refrigerated centrifuge, pH meter, universal food processor, water bath, colorimeter, torsion gelometer, dynamic rheometer, viscometer, texture analyzer, gas chromatograph, and other analytical equipment. Dr. Jaczynski has a sufficient laboratory space where the new equipment (i.e., two bio-reactors with chillers, two continuous centrifuges, and homogenizer) will be installed and set up. Dr. Jaczynski’s existing equipment will be used for laboratory analyses necessary to conduct the proposed research.

Facilities of the College of Engineering and Mineral Resources contains in excess of 5000 ft² of laboratory space. Analytical equipment includes: one atomic absorption spectrophotometer; three gas chromatographs with multiple detector arrays (PID and FID) and a purge and trap apparatus; two total organic carbon analyzers; three automated pH titrators; one scanning electron microscope; and one UV-visual light absorbance spectrophotometer. The National Research Center for Coal and Energy (NRCCE) located adjacent to the engineering facilities will conduct water quality analyses. The laboratory is EPA certified for performing analysis under the National Pollutant Discharge Elimination System of the Clean Water Act. All analytical methods are EPA approved and have a standard Quality Assurance/Quality Control protocol.

Proposed work will take place at two pilot scale raceway systems. Each system is composed of HFRP material with four levels of paired units, each 30 feet long capable of maintaining approximately 1000 lb of fish. Combined, the systems are supplied with about 1000 gallons/minute, and have a total of 16 experimental units. The facility at Dogwood Lake, approximately 15 miles west of WVU main campus is supplied with treated mine water. The facility at Reymann Memorial Farm is part of the WV Agricultural Experiment Station near Wardensville, WV and is fed by spring water.

The raceway system at the Reymann Memorial Farm in Wardensville, WV, was selected to study nutrient recovery with watercress. The spring feeding the facility naturally supports watercress and is representative of small springs found throughout the region. Minimizing nitrogen in the Potomac River headwaters is an important consideration for growers in this area. A temporary building has been installed enclosing both the raceways and additional space where controlled experiments may be performed. Power is available at the site.

Most experiments described in this proposal will take a year to complete. Not all components will begin at the same time, however and may depend on work presently underway to be completed. Analysis and technology transfer efforts will require additional time such that projects are expected to be complete in two years.

Adler, P.R., J. K. Harper, F. Takeda, E.M. Wade and S.T. Summerfelt. 2000. Economic evaluation of hydroponics and other treatment options for phosphorous removal in aquaculture effluent. HortScience 35:993-999.

Adler, P.R., F. Takeda, D.M. Glenn, E.M. Wade, S.T. Summerfelt and J.K. Harper. 1996. Nutrient removal: ecological process sows a cost-saving idea for enhancing water quality. Water Environment and Technology 8:23-24.

Agarwal, B.D., and Broutman, L.J. (1990). Analysis and Performance of Fiber Composites, 2^nd Edition, John Wiley & Sons, Inc. New York, NY. Pp. 37-40.

American Public Health Association, 1998. Standard Methods for the Analysis of Water and Wastewater, 20^th edition. American Public Health Association, Washington, DC.

Anderson, D.M., P.M. Gilbert and J.M. Burkholder. 2002. Harmful algal blooms and eutrophication: nutrient sources,
composition and consequences. Estuaries 25: 704-726.

Barrows, F.T. and R.W. Hardy, Nutrition and Feeding, in Fish Hatchery Management, 2^nd ed., G.A. Wedemeyer, editor, American Fisheries Society, Bethesda, MD (2001)

Choi YJ, Park JW. 2000. Feasibility study of new acid-aided surimi processing method for enzyme-laden Pacific whiting. Abstract # 51A-4. Presented at the Institute of Food Technologists Annual Meeting (Dallas, TX). June 10-13.

Davalos, J.F., and Chen, A. (2005). “A Solution Including Skin Effect for Stiffness and Stress Field of Sandwich
Honeycomb Core,” International Journal of Solids and Structures. 42: 2711-2739.

Hultin HO, Kelleher SD. 1999. Process for isolating a protein composition from a muscle source and protein composition.
US Patent No. 6,005,073. Issues Dec. 21.

Klontz, G.W. 1991 Manual for Rainbow Trout Production on the Family Owned Farm, Nelson and Sons, Inc., Murray,
UT (1991)

Lanier, T.C. 2000. Surimi gelation chemistry. In: Park JW, editor. Surimi and surimi seafood. New York: Marcel Dekker. p 237.

Lee, H., and TC Lanier. 1995. The role of covalent crosslinking in the texturizing of the muscle proteins sols. J Muscle Foods 6:125-138.

Loch, D. D., J. L. West, and D. G. Perlmutter. 1996. The effect of trout farm effluent on the taxa richness of benthic macroinvertebrates. Aquaculture 147:37-55.

Lopez-Anido, R., Davalos, J.F., and Barbero, E.J. (1995). Experimental Evaluation of Stiffness of Laminated Composite
Beam Elements under Flexure, Journal of Reinforced Plastics and Composites, 14, 349-361.

Makkapati, S. 1994. Compressive Strength of Pultruded Structural Shapes, Master’s Thesis, West Virginia University.

Piper, R.G., I.B. McElwain, L.E. Orme, J.P. McCraren, L.G. Fowler, and J.R. Leonard, 1982. Fish Hatchery
Management, US Department of the Interior, Fish and Wildlife Service, Washington, D. C.

Rakocy, J.E., R.C.Shultz, and D.S. Bailey. 2000. Commercial Aquaponics for the Caribbean. Proceedings of the Gulf and Caribbean Fisheries Institute 51: 353-364.

Selong, J. H. and L. A. Helfrich. 1998. Impacts of trout culture effluent on water quality and biotic communities in Virginia headwater streams. Progressive Fish-Culturist 60:247-262.

Semmens, K., 2005. Assessing Three Stocking Densities for the Production of Female Green Sunfish x Male Bluegill
Sunfish Hybrids in West Virginia. Abstract and presentation at the World Aquaculture Society National Conference &
Exposition, Aquaculture America 2005, January 17-20, New Orleans, LA.

Soderberg, R.W., Flowing Water Fish Culture, CRC Press LLC, Boca Raton, FL (1995)

Srinivasan, D., 1996. Amino acids, peptides, and proteins. In: Fennema OR, editor. Food Chemistry 3^rd ed. New York: Marcel Dekker. p 322.

USEPA, 1993. Guidance for Assessing Chemical Containment Data for Use in Fish Advisories, Vol. I. Fish Sampling
and Analysis (EPA 823-R-93-002). U.S. Environmental Protection Agency, Washington, D.C.

USEPA, 1998. Test Methods For Evaluating Solid Wastes, Physical/Chemical Methods, Revision 5 (EPA SW8-46).
U.S. Environmental Protection Agency, Washington, D.C.

Vantaram A., J.F. Davalos, J. Robinson, and J.D. Plunkett. “Honeycomb Fiber-Reinforced Polymer Sandwich
     Construction for Development and Implementation of Fish Raceway Systems,” Proceedings of the ASC 17^th Annual
    Technical Conference, American Society of Composites (ASC), Purdue University, West Lafayette, Indiana, Oct.
    21-23, 2002 (Paper#081).

Viadero, R., A. Tierney, and K. Semmens, 2004. “Use of Treated Mine Water for Rainbow Trout (Oncorhynchus
mykiss) Culture – A Production Scale Assessment,” Aquacultural Engineering, 31 (3-4), 319-336.

Viadero, R., and A. Tierney, 2003. Use Of Treated Mine Water for Rainbow Trout (Oncorhynchus mykiss) Culture – A
Preliminary Assessment. Aquacultural Engineering, 29 (1-2) 43-56.

Yongsawatdigul, J., and J.W. Park, 2001. Gelation characteristics of alkaline and acid solubilization of fish muscle proteins. Abstract # 100-1. Presented at the IFT annual meeting (New Orleans, LA). June 23-27.

Kenneth J. Semmens. Dr. Semmens, is State Extension Specialist for Aquaculture and specializes in aquaculture with twenty years of experience producing and marketing a wide variety of warm, cool and coldwater fish species. He holds a joint appointment with the Cooperative Extension Service and the West Virginia Agricultural and Forestry Experiment Station. He will oversee the execution of the project’s activities, assist the co-investigators with problems and issues that arise, facilitate resource allocation and integrate project activities in support of the aquaculture industry in West Virginia. He will also direct Technology Transfer activities (objective 6).

Karen M. Buzby is a postdoctoral fellow in the Department of Civil and Environmental Engineering at West Virginia University. She will collaborate with Dr. Todd West in objective 4, nutrient removal from trout raceway effluent. She will focus on water chemistry.

Julio Davalos is C.W. Benedum Distinguished Teaching Professor, Department of Civil and Environmental Engineering, College of Engineering and Mineral Resources, West Virginia University. His primary research interests are analytical and applied mechanics, characterization of wood and fiber-reinforced polymer composites, structural and bridge engineering, and effective teaching methods. Dr. Davalos has produced design manuals and taught courses on composite materials. He will lead the task developing cost competitive HFRP tanks.

Jacek Jaczynski is an assistant professor of food science in the Division of Animal and Veterinary Sciences at West Virginia University. His interests are aquatic foods and food safety. He will lead task 5, Protein and Lipid recovery from Fish Processing By-products.

Rodney Kiser, is a Research Assistant II with the WVU Cooperative Extension Service. His duties focus primarily on assisting landowners and producers utilizing farm raised fish in recreation.

Daniel Miller, M.S., is Research Associate in the Agricultural and Resource Economics Program. Mr. Miller’s work experience emphasizes the biological and production aspects of aquaculture and fisheries. He has served as a manager of and consultant on several aquaculture‑related projects in various parts of the U.S. and overseas. His work on the project will focus on technology transfer particularly development of the mine water resource for commercial production of salmonids.

Aislinn E. Tierney, is an associate engineering scientist in the department of Civil and Environmental Engineering. She coordinates water quality and effluent analysis at the WVU raceway facility utilizing treated mine water and assists Dr. Viadero with research on the engineering aspects at two mine water aquaculture facilities.

Richard Turton, Professor of Chemical Engineering will supervise the development and verification of the software simulation tools to simulate fish growth in raceway systems (objective 3). Dr. Turton has extensive experience in modeling and software development and was central to the development of the single tank raceway software.

Roger C. Viadero, Jr., Assistant Professor of Civil and Environmental Engineering will direct research associated with objectives 1 (Production of Four Fish Species through Year-Round Operation of a Flowing Water System Utilizing Treated Mine Water)

and objective 4 (Nutrient Removal from trout raceway effluent). Dr. Viadero has led research on utilizing mine water for trout production and the engineering aspects of water treatment in recirculating aquaculture systems used to raise yellow perch.

Yin-Han Wang, MS graduate student in the Department of Chemical Engineering responsible for software development will assist Dr. Turton on objective 3.

Todd West, Assistant Professor of Horticulture, will collaborate with Dr. Viadero and Dr. Buzby on objective 4 - Nutrient Removal from trout raceway effluent.