Progress 07/01/22 to 02/28/23

Outputs
Target Audience:AST markets several hundred new bead filters each year into the aquaculture, aquaponic, R & D laboratories, zoo, food distribution and koi pond markets each year, and most of these filters go to individuals with little or no experience acclimating biofilters. Our company is chronically getting calls from customers that are frustrated during acclimation with the persistent accumulation of ammonia then nitrite. They are mystified by the mathematics of bacterial reproduction which typically resolves a long-term accumulation almost overnight. Untold numbers of tropical fish, ornamental koi, salamanders, baitfish, shrimp, crawfish, crabs, and lobsters fall prey to poor acclimation. In the extreme, the process can take 4-5 months to complete (typically associated with cooler marine applications). Elimination of this problem would dramatically enhance the perceived value of our entire aquatic line which ranges in size from 3 liters to 100 cubic feet of media. The second market for the start-up beads would be shock loading mitigation. Several industries have their RAS cyclically loaded. Perhaps the worst of these are the blue crab shedding systems that are dependent on temperature driven molting cycles. In the cold spring, virtually no molting crabs are available, then a warm front passes and the unacclimated systems are overwhelmed with a surplus of pre-molt crabs. Particularly sensitive to low nitrite levels, molting losses are extremely high as the owners of these land-based systems wait for the slow acclimation process. Baitfish systems, typically empty by Saturday noon, are shocked with a fresh supply Thursday morning. Similarly, live lobster distribution lines are cyclically shocked. There are sophisticated means of dealing with this problem (like drip feeding ammonia), but only a small percentage of our clients have the expertise to master these approaches. These shock markets represent a potential base for reoccurring sales. Units could be surcharged with acclimation beads that would help absorb the shock by assimilation. It is likely that the start-up beads will be marketed directly to our long-term customer base by offering the start-up beads on Amazon. AST now successfully offers several filter models and aquaponic systems via Amazon. Moderate quantities (perhaps in 1 to 5-pound quantities) of start-up beads packaged and available with free shipping would be well received. Industries that will benefit from the use of this technology include commercial finfish production facilities, catfish, trout, baitfish, marine shrimp, striped bass, sea bass, flounder, and other commodity food stock. The live seafood distribution infrastructure includes a large number of marine and freshwater RAS holding animals such as lobsters, tilapia, blue crabs, sea bass, and other aquatic animals for display at point of sale, for holding at distribution nodes, and for purging at the initial point of landing. Research facilities including those holding aquatic animals for biomedical studies, for genetics studies, and more generic university studies would become target markets for a start-up bead. The start-up beads would also benefit the ornamental fish industry as they try to produce, distribute, sell, and eventually enjoy the wide diversity of aquatic animals distributed throughout the U.S. Finally, the public aquaria display industry, operated by the State, the Federal Government, communities, zoos, and private industries typically have numerous support systems and/or RAS that would benefit from the ability to acclimate without losing animals. Changes/Problems:There were no fundamental changes in our strategy. The experimental work was challenging because of the timeframe and the process of discovery. The research team knows a lot more now than a year ago. The state-of-the-art has been advanced. Some of the issues follow. Initial BOD testing failed to quantify the relative biodegradability of the biodegradable plastics evaluated for the assimilation bead, so this preliminary approach was abandoned. The researchers were able to successfully coat expanded glass cores with a variety of bioplastics for comparative tests, but given the timeframe, were not able to produce beads of consistent diameter for direct comparative quantitative tests. So, it was opted to modify the test biological filters to support either floating or sinking beads in comparative tests. Differences in bead diameter were tolerated. Replicated biological testing was successfully completed. These tests confirmed the feasibility of the "start-up" bead, but once again failed to quantify a significant difference in performance. The researchers had assumed that the rate of assimilation would be driven by the carbon content of the assimilation beads, but in the detailed biological tests, all bead compositions performed identically leading to the conclusion that a secondary process (likely, ammonia or oxygen diffusion through the biofilm) was limiting the rate. Testing confirmed biofilm management (backflushing) was an important element in improving rates. So, ongoing work was more focused on bead management, and acclimation test results improved. This marine and freshwater data demonstrate the potential of start-up bead concept, despite problems of biofouling in the experimental start-up bead compartment. The chamber at the top of the filters supporting sinking assimilation beads were subject to biofouling due to the limited space for washing. This problem reinforced the conclusion that start-up beads should float, so they can be deployed dispersed in a bead bed. What opportunities for training and professional development has the project provided?Over the course of this research project, AST has employed 6 undergraduate students from Louisiana State University from various engineering and marketing disciplines. The types of things the students have been taught are as follows: Fundamental lab protocol (emphasizing lab safety) Proper methods of taking, handling, and storing water samples Methods of measuring water quality parameters (total suspended solids, ammonia, nitrite, nitrate, dissolved oxygen, pH, alkalinity, temperature, salinity, sulfide, and turbidity) held to academic research standards as well as the meaning behind the measurements. They were also shown how to utilize Standard Methods to figure out how to measure various water quality parameters How to compile and interpret data Strategies and techniques used to develop a targeted market YouTube video marketing techniques How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? All project objectives were met. Three compositions were demonstrated as satisfactory for use in assimilation beads. One of the three was favored because of its lower cost. This composition displayed a specific density less than 1 indicating its compatibility with the floating bead filter format. Further, improvement in performance and reduction in cost can be expected as work progresses. The Phase I effort was undertaken under the assumption that the carbon content and its availability would limit the assimilation rate of the various particulate carbon bases. The preliminary review (Phase I; Task 1) of a wide variety of biopolymers and bioplastics displayed promising aerobic decay rates. Secondary factors such as melting point and density were then considered to identify candidates for evaluation. BOD5 evaluations followed procedures described in the scientific literature, but the research team quickly found that secondary factors prevented a clear identification of the best candidates. The research team was not able to identify an in-house production technique that would produce a dimensionally controlled bead that would facilitate direct BOD5 comparisons. However, having run over 250 BOD5 tests, three compositions showed promising biodegradability in the BOD5 testing and were advanced for more detailed experimentation. The three base compounds were selected: a high temperature bioplastic, a low temperature bioplastic, and a low temperature biopolymer. The three base compounds displayed promising biodegradability, had literature support, and were commercially available in a pelleted form. Each compound behaved similarly on a per trial basis, but the research team concluded that factors other than biodegradability were controlling outcomes. A mass balance model (Phase I; Task 2) was developed in Stellatm modeling environment using approximate nitrogen assimilation rates from the BOD5 tests. The model was used to assess the estimated carrying capacity that 0.1 liters of start-up beads could support. The information from Phase I; Task 2 was used to estimate the number of animals that could be used in the proof-of-concept tests (Phase I; Task 4). There were three studies targeted for the (Phase I; Task 4): a) a marine study, b) a freshwater study, and c) a marine nitrate enrichment study. An 8 x 16-ft insulated constant temperature room was fabricated by the AST production staff. The room was equipped with air conditioning and heaters. Twelve systems were constructed using one Nano6000 PolyGeyser® filter, one 30-gallon tank, one air stone, and one pump. A set of aluminum stands was fabricated to support the tanks. Two of our selected bases sank, and one floated. To equalize the comparison, the Nano 6000 PolyGeysers® had to be modified to create a double screened chamber above the bead bed. This chamber was filled with a tenth of a liter of start-up beads in nine of the test filters and a tenth of a liter of low-density polyethylene in three of the control filters. All twelve were then filled with a liter of low-density polyethylene in the normal bead bed chamber. This gave all twelve filters a total of 110% beads. Under normal circulation, water coming from the tank first passed through the EN media then through the start-up media. The start-up bead bases and controls were always tested in triplicate. The first experiment (Phase I; Task 2a) was conducted with the red swamp crayfish, Procambarus clarkia, in freshwater. In these experiments, 75 grams of crawfish (six individuals) were added to each tank. The crawfish were fed 1.5 grams of food per day. The procedures followed were identical to the marine studies. Peak TAN for the start-up bead treatments ranged from 30-50% of the control's peak. The ammonia acclimation occurred on the 10th day for the control and on the 12th for the start-up bead treatments. There was no initial lag in start-up bead treatments. Impacts on ammonia by the treatments were clear by the 2nd day. For the Phase I, Objective 2b, the flow was set to one gallon per minute on all tanks. The twelve systems were filled with tap water and salted to 20 ppt. They were bleached at 2 ppm-Cl2 for two hours to kill all nitrifiers and then neutralized using sodium thiosulfate. 80-90 grams of shrimp (three large individuals) were added to each tank. The shrimp were fed 1 gram of food per day. Ammonia, pH, and nitrate were initially measured, and ammonia was monitored throughout the study. Nitrite and nitrate concentration were measured as ammonia began to drop. The systems were backwashed, and sludge was drained once per day. The temperature was kept at 30 oC. It was clear that biofouling of the beads was a major controlling factor as heavily biofouled bead beds that had gelled together could be observed. Animal loading was limited to minimize biofouling and allow the comparison of bases to proceed. The shrimp benefitted from this decision as none died from exposure to the controls peak TAN concentration (1-2 mg-N/L) and the shrimp even molted without difficulty throughout the marine studies. All tested bases worked well in the marine study. The peak ammonia concentration with the bases ranged from about 20-40 percent of the controls. All systems appeared to acclimate by the 11th day as evidenced by appearance of nitrite in the systems. The rebounding of the ammonia curves after the 11th day is caused by organic biofouling of the start-up bead compartment. A detailed ANOVA analysis confirmed there were no significant differences between the start-up bead bases in either the freshwater or the marine studies. The third experiment (Phase I; Task4c) was a marine, elevated nitrate study conducted to examine how the bacteria acted in an environment with excess nitrate. High nitrate levels are typically the result of reusing water in production for more than a few months. It was hypothesized that the heterotrophic bacteria could preferentially consume nitrate instead of ammonia. This study was undertaken to confirm that the start-up bead concept would work in the presence of nitrates. In this experiment, nine systems were employed. Using Base #3 at 10 percent, 3 systems were dosed with 0, 50, and 100 mg-N/L nitrate. Otherwise, the experimental procedures were identical to the first marine experiment (Phase I; Task 4a). Despite the presence of nitrate, the start-up beads were still able to keep ammonia concentrations down below 1 ppm. Peak ammonia concentrations ranged from 0.4 to 0.6 (20-30 percent of the control). The bacteria preferentially selected ammonia as a nitrogen source for assimilation over nitrate. There was no statistically identifiable difference between the nitrate doses. These results indicate that start-up beads will be able to provide relief for ammonia buildup in both new systems and in reacclimating old systems where the previous use may have burdened water with excess nitrate concentrations. The Phase I Task 5 effort focused on the relative cost of the selected compositions (bases). Since there is little net difference in the nitrogen assimilation capacity of the selected bases, this analysis was reduced to a cost per kg analysis. Bulk prices of Base #1 are currently estimated at $5.5/kg. Base #2, which is widely marketed, was estimated at $31/kg. The Base #3 that was tested in Task 4 was estimated at $4.85/kg. Thus, at this point, Base #3 appears to be the least expensive option. The cost to remove one kg-N per base is as follows: Base #1: $67.61/kg-N, Base #2: $252/kg-N, Base #3: $30.7/kg-N. Thus, Base #3 was considered the best of the three tested. The project successfully identified three bead compositions that work. One of the beads already floats (assuring compatibility with the floating bead bed) and is already available as beads. This facilitates off-facility beta testing and could support initial marketing efforts. So, the Phase I effort was successful.

Publications