Progress 07/01/24 to 02/28/25

Outputs
Target Audience:Theprimary audiences for the project's efforts are 1) aquaculture research facilities, (e.g., University of Southern Mississippi's Thad Cochran Marine Aquaculture Center, North Carolina State's Marine Aquaculture Research Center, and Virginia Tech's Virginia Seafood Agricultural Research and Extension Center), and 2) industrial aquaculture facilities, such as The Freshwater Institute, Blue Ridge Aquaculture Inc., and Superior Fresh, LLC. Outreach efforts to these groups include sharing updates regarding Phase I results and discussion of technology needs and strategies for approaching field implementation . Research facilities such as the Freshwater Institute are at the forefront of developing data, methods, and technologies that directly benefit the aquaculture industry through their extensive industry networks. Other research facilities such as North Carolina State University disseminate findings through publications, websites, case studies, workshops, conferences and extension programs to work directly with farmers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Professional development was advanced primarily through the design and execution of experiments and communication of experimental results. The team developed protocols, ran experiments, and gathered data to meet project objectives of determining reaction kinetics and developing continuous-flow prototypes for total inorganic nitrogen (TIN) treatment in synthetic and real recirculating aquaculture system (RAS) effluents. Written reports and formal data analysis were completed for Phase I, furthering the company's understanding of biocatalysts developed for the aquaculture use case. Communication with outside organizations allowed the team to secure actual RAS waste for bench-scale testing, which is of high interest to industry professionals who are utilizing RAS for either research purposes or human consumption. Additionally, it allowed team members to practice presenting technical data, explore the industry's research and technology landscape, identifytarget regions and applications, grow the company's and team's network, and generate interest in the technology. This project also provided multiple technical training opportunities for the project team. First, organisms previously unfamiliar to Microvi's research team were utilized over the course of the project, which provided training opportunities on culturing of these new organisms. Testing salty conditions in the bench-scale experiments provided experience in preparation and testing of saline bioreactors. This included introducing the use of a refractometer and a total dissolved solids (TDS) meter. How have the results been disseminated to communities of interest? A summary of Phase I results has been shared and discussed with both academic and industry stakeholders through email and virtual meetings. These conversations were used to support Phase I testing and Phase II planning and potential partnerships. Following the close of Phase I, the project team will share project results via peer-reviewed publication and conference abstracts and presentations. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: An organism which can denitrify in saline environments (halotolerant denitrifier) and one that can oxidize ammonia in saline conditions (marine nitrifier) were sourced from DSMZ and ATCC, respectively. Cultivation of ATCC 19707 Nitrosococcus oceani, was successful and reserve glycerol stocks were prepared. However, the growth rate was considerably slow such that not enough biomass could be prepared. Similarly, efforts in cultivation of DSM 10037, Virgibacillus halodentrificans, did not yield enough biomass for Objectives 2 and 3. Alternative organism P. stutzeri was selected and grown. The resulting biocatalyst composites were then subject to denitrification studies under various salinity levels, while a nitrifying biocatalyst containing heterotrophic aerobic bacteria were selected for testing of nitrification under saline conditions. Objective 2: Specific denitrification kinetics were investigated by comparing two denitrifying biocatalysts containing P. stutzeri and one containing a standard denitrifier for non-saline applications. The composites were acclimated repeatedly in parallel using a nutrient-rich media spiked with 50 mgN/L nitrate and sodium acetate as the sole carbon source. Once the denitrifying activity was verified, the large portion was split into four equal parts for testing in denitrifying medium. Increasing levels of salinity supplied as sodium chloride were all tested in parallel. In total, eight flask reactors were tested, each type of biocatalyst was subject to three batch tests under either a 0%, 1%, 2%, and 4% NaCl denitrifying medium. Tests were repeated three times to determine the specific reaction kinetics under the starting condition with 80 mg-N/L nitrate-Nitrogen. The first-order rate constants for each biocatalyst under four conditions were determined. All results indicated a trend in decreasing rate constant as salinity increased. The only instance of statistically significant difference between the two denitrifying biocatalyst was for the 0% NaCl medium, which indicated that the existing biocatalyst outperformed the newer iteration with P. stutzeri. It was hypothesized that P. stutzeri biocatalyst performance could be improved by using an alternative carbon source such as citrate, but this was left out from testing protocols due to the impracticality of using citrate for full-scale applications. The cases for using a standard denitrifier compared to P. stutzeri for aquacultural applications are kinetics as related to denitrification efficiency, minimum COD:N ratio for performance, ability to utilize dissolved carbon in actual waste streams, and long-term salinity-tolerance. Objective 3: Lab-based bench-scale units were designed and tested under different configurations leading up to their optimized final form. Two parallel processes for the treatment of synthetic aquaculture wastes based on continuous-flow stirred-tank reactors (CSTRs) were built and operated on the bench: One system contained a denitrifying CSTR utilizing P. stutzeri biocatalyst followed by a CSTR containing an aerobic nitrifying biocatalysts. Rather than relying on a pump in between reactors, the outfall from the anoxic denitrifying CSTR was gravity-fed into the second, aerobic CSTR. The second system consisted of a denitrifying CSTR utilizing denitrifying biocatalyst followed by the aerobic nitrifying biocatalyst CSTR. Anoxic CSTRs were dosed continuously with acetic acid carbon source (electron donor) and nutrient (orthophosphate, 100:2 COD:P ratio) from a concentrated feed to maintain a 1.5 times theoretical demand for the given nitrate-N load. Hydraulic retention times were approximately 7.5 and 7.8 hours for anoxic and aerobic CSTRs, respectively. Influent was a synthetic RAS wastewater containing both 80 mg-N/L nitrate and 10 mg-N/L ammonium. The elimination capacity was measured each day by collecting grab samples from influent, anoxic effluent, and aerobic (final) effluents and measuring ammonia-N, nitrite-N, nitrate-N (combined these represent TIN), and soluble COD. Baseline data for the non-saline conditions was collected first, and then the following conditions were tested: 1, 2, and 4% salinity supplied as NaCl. In between 0% and 1% test conditions, a brackish (0.5% saline) actual RAS effluent was used as influent for both systems. In between the 2% and 4% saline conditions, a real mariculture RAS effluent having 2.6% salinity was used as the influent for both systems. Bench units were effective to treat Total Inorganic Nitrogen (TIN) in both synthetic and actual recirculating aquaculture systems' effluents. For P. stutzeri and nitrifying biocatalyst-filled CSTRs in series, the operation with 0, 1, 2, and 4% NaCl synthetic RAS wastewater resulted in 60.8%, 55.5%, 59.3% and 77.8% average TIN removal efficiency, respectively. The system responded dynamically to new salinity conditions, and therefore some residual nitrite-N was detected for grab samples with improved suppression of nitrite-N buildup during after longer duration of the operation. Significantly, residual ammonium-N exceeded 2 mg-N/L for select grab samples under the 1, 2 and 4% salinity test conditions but not for the non-saline baseline condition. For the denitrifying and nitrifying biocatalyst CSTRs in series, the operation with 0, 1, 2, and 4% NaCl synthetic RAS wastewater resulted in 50.5%, 62.6%, 60.0% and 70.1% average TIN removal efficiency, respectively. For saline conditions, residual nitrite-N was substantial for this treatment process (effluent nitrite-N>3 mg-N/L), indicative of incomplete denitrification. Given the toxicity of nitrite-N to many aquatic species, this result points to the favorability of using P. stutzeri biocatalyst for anoxic denitrification especially for the saline use-case. Research was conducted to understand the potential TIN discharge limits at various sites, or percent TIN or nitrate-N reduction that would be needed for the water to be effectively reused. This guided the experimentation and assessment of bench bioreactors and helped compare results with those of conventional treatment systems. Further research indicated strict limitations on other water quality parameters (e.g., nitrite-N, ammonia-N) that are of high concern during cultivation of fish and aquatic species; this helped to highlight areas where having flexible process control may help biocatalyst performance, or situations when advanced process instrumentation (e.g., nitrite-N online analyzer) could be implemented as well. A preliminary technoeconomic analysis (TEA) was conducted to compare other nitrifying biofilter technologies to Microvi's biocatalysts under relevant conditions. Recirculating Aquaculture System (RAS)Technologies(2006) by N. Timmons, M.B. Timmons, and J. Ebeling compares the cost of a rotating biological contactor, trickling biofilter, bead filter, and fluidized-sand biofilter for a 0.8 million gallons per day (3000 m3/day) RAS producing tilapia. The Microvi system would require two 11,450-gallon tanks with ~27,000 lbs of biocatalyst media and would cost approximately $500,000 (including system and media), which is less than the median and average (~$680,000 and ~$730,000, respectively in 2025 dollars) of the options described in Timmons et al, 2006 and demonstrating the Microvi technology's cost effectiveness.

Publications