ELUCIDATING NUTRIENT LOADS AND BIOAVAILABILITY TO CYANOBACTERIA IN CATFISH PONDS FOR SUSTAINABLE AQUACULTURE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1032233
• Grant No.: 2024-67019-42686
• Cumulative Award Amt.: $444,559.00
• Proposal No.: 2023-09881
• Project Start Date: Jul 1, 2024
• Project End Date: Jun 30, 2027
• Grant Year: 2024
• Program Code: [A1411]- Foundational Program: Agricultural Water Science

Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849

Performing Department
(N/A)

Non Technical Summary
An actionable understanding of nutrient load and bioavailability to cyanobacteria in freshwater systems is fundamental to managing water quality and mitigating harmful algal blooms (HABs), especially under global warming pressures. However, the decades-long practices for water quality analyses by USDA, as well as federal and state regulatory agencies (e.g., USEPA), depend heavily on the cutoff that separates nutrient fluxes between particles and the dissolved phase using a nominal 450-nm pore-size filter membrane. The obtained dissolved phase (i.e., <450 nm) includes small colloids (<450-100 nm), nanoparticles (<100-1 nm), and the truly dissolved phase (<1 nm), which significantly overestimates nutrient load in the truly dissolved phase. The long-term inaccurate knowledge of nutrient load and bioavailability in different size fractions precludes us from formulating effective plans for nutrient management and water quality restoration. The goal of the proposed project is to accurately reevaluate the 'real contribution' of different size fractions on nutrient load and bioavailability to cyanobacteria in economically important catfish aquaculture ponds in Alabama under a warmer climate. The findings obtained from the proposed project will enable USDA, regulatory and state agencies, catfish producers, and other aquaculture stakeholders to improve current best management practices (BMPs) for the forecasting and mitigation of cyanobacterial HABs using cost-effective engineered solutions (e.g., engineered biochars), contributing towards more sustainable aquacultural productivity and profitability in a warmer climate.

Animal Health Component

20%

Research Effort Categories

Basic

70%

Applied

20%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
102	0210	2000	70%
133	0810	2020	30%

Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships; 133 - Pollution Prevention and Mitigation;

Subject Of Investigation
0810 - Finfish; 0210 - Water resources;

Field Of Science
2020 - Engineering; 2000 - Chemistry;

Keywords

nutrients

harmful algal blooms

restoration

sustainable aquaculture

water quality management

Goals / Objectives
An actionable understanding of nutrients--phosphorus (P) and nitrogen (N)--load and bioavailability to cyanobacteria in freshwater systems is vital for water quality management and harmful algal bloom (HAB) mitigation, especially under global warming pressures. The goal of this project is to accurately reevaluate the 'real contribution' of different size fractions of particles on nutrients (P and N) load and bioavailability to cyanobacteria in economically important catfish production ponds in Alabama. This project has four main objectives:Objective 1: Collect bimonthly water samples from 21 catfish aquaculture ponds in Alabama and physically separate the collected water samples into six-size fractions, with two Tasks:Task 1: Bimonthly water sampling of 21 catfish aquaculture ponds in AlabamaTask 2: New particle size separation using sequential centrifugation and ultrafiltrationObjective 2: Chemically quantify nutrients (P & N) concentration and speciation, along with key algal-related water quality parameters (algal pigments, algal toxins, and off-flavors and odors) in the six size fractions to relate with water temperature (i.e., seasonal change), with two Tasks:Task 3: Re-analyzing concentrations of nutrients and algal parameters in different size fractionsTask 4: Re-analyzing relationships of nutrients with algal parameters in different size fractionsObjective 3: Biologically test the bioavailability of nutrients in the six size fractions under different temperatures (mimic climate change) to three cyanobacteria (Microcystis, Cylindrospermopsis, and Dolichospermum) isolated from aquaculture ponds, with three Tasks:Task 5: Colloidal stability, aggregation, and sedimentation of particles in different size fractionsTask 6: Bioavailability tests to three cyanobacteria grown individually in a warmer climateTask 7: Bioavailability tests to three mixed cyanobacteria in a warmer climateObjective 4: Develop and implement cost-effective solutions (e.g., engineered biochars) for water quality management and HAB mitigation in aquaculture ponds for sustainable aquaculture, with one Task:Task 8: Cost-effective biochars for nutrient control and HAB mitigation in aquaculture ponds.The findings will enable USDA, regulatory agencies, and aquaculture stakeholders to adopt best management practices (BMPs) and implement cost-effective solutions (e.g., engineered biochars) for the mitigation of HABs, contributing toward more sustainable aquaculture productivity and profitability for a growing world population.

Project Methods
This project will involve (1) bimonthly water sampling from catfish aquaculture ponds in West Alabama; (2) particle size separation of the collected water samples; (3) analyses of water quality parameters, including nutrients(phosphorus and nitrogen) and algal-related parameters; (4) nutrient bioavailability tests to threecyanobacteria,and (5) test and use engineered biochars for removing nutrients from ponds.1. Bimonthly water sampling from catfish aquaculture ponds in west Alabama.Water samples will be collected bimonthly to identify temporal variations of particles on nutrient load in aquaculture pond systems. Water sampling procedures are described below: water samples will be collected using a rigid plastic tube that integrates water samples from the surface to a depth of 0.5 m. The collected water samples will then be stored in clean plastic containers in coolers until processed in the lab (<6 h holding time). Basic water quality parameters, including T, pH, dissolved oxygen, electrical conductivity, and photosynthetically active radiation, will be analyzed in situ using a YSI Multi-Sensor and a Li-Cor Light-Meter, respectively. A field sensor, bbe Moldaenke PhycoProbe, will also be used to discriminate ambient phytoplankton abundance and taxa rapidly. The collected water samples will be processed in the lab for various water quality parameter analyses (e.g., chlorophyll, phycocyanin, and others).2. Particle size separation of the collected water samples.A new approach based on centrifugationand ultrafiltrationwill be used to obtain the large particles (>1,000 nm), large colloids (1,000-450 nm), small colloids (<450-100 nm), large NPs (<100-50 nm), small NPs (<50-1 nm), and the truly dissolved phase (<1 nm). To achieve this, the ThermoScientific Sorvall LYNX 4000 superspeed centrifuge (4 L capacity with the highest speed of 68,905×g) will be used to separate water samples into large particles (>1,000 nm), large colloids (1,000-450 nm), small colloids (<450-100 nm), and large NPs (<100-50 nm) based on the Stokes' Law of particle settling (by varying the centrifuge speed and time duration). The supernatants, after sequential centrifugation, will then be used for the ultrafiltration (400 mL capacity). Prior to the ultrafiltration, all stirred cell units will be acid-cleaned, and the 10 kDa cellulose membranes (pore size ≈ 1 nm; Millipore Sigma) will also be cleaned with 0.05 M NaOH and ultrapure water.The routine protocol of using a 450 nm filter membrane by USDA and USEPA will also be conducted in parallel to compare the differences of two particle size separation approaches, including nutrient load, speciation, and relationship analyses with key algal-related water quality parameters.All separated particles with different sizes will be quantified gravimetrically on a Mettler Toledo microbalance (0.1 µg accuracy), which will then be stored in a refrigerator at 4 °C for later use.3. Analyses of water quality parameters (P, N, and algal-related parameters).The Hedley's extraction methodwill be used to separate P pools into DI-P, NaHCO3-P, NaOH-P, HNO3-P, and residual-P in different size fractions.Inorganic P (Pi)concentration in these extracted P pools will be quantified by the phosphomolybdate blue method.Total P (TP)concentration will be determined after persulfate digestion.Organic P (Po)concentration will be calculated by subtracting Pifrom TP.Theliquid31P NMR spectroscopywill be used to characterize different P speciation of particles in different size fractions on the Bruker UltraShield 600 MHz NMR spectrometer at 129.5 Hz for31P at AU. Besides P, N, including total N (TN) and nitrate (NO3-) concentrations in different size fractions, will also be analyzed, following the procedures well-operated in Wilson's lab.Key algal-related water quality parameters, including algal pigments (chlorophyll for all algae and phycocyanin for cyanobacteria), algal toxin microcystin, and two common taste and odor compounds, MIB and geosmin, will be monitored, following Wilson's work.Briefly, chlorophyll and phycocyanin concentrations (µg/L) will be determined by fluorometric analysis on a Turner Designs Trilogy fluorometer (non?acidification chlorophyll moduleand orange module,respectively) after extraction using 90% aqueous ethanol or 50 mM phosphate buffer, respectively. Microcystin and cylindrospermopsin concentrations (µg/L) will be quantified via enzyme-linked immunosorbent assay (ELISA) after extraction with acidified aqueous methanol.The MIB and geosmin concentrations (ng/L) will be analyzed on a gas chromatography/mass spectrometry (GC/MS) after solid phase microextraction.4.Nutrient bioavailability tests to three cyanobacteria.Bioavailability tests will focus onMicrocystis,Cylindrospermopsis, andDolichospermum, and all 3 cyanobacteria are currently ready for use. The cyanobacterial strains will be cultured in the I-36VL Percival incubator under a 12 h/12 h day/night cycle with illumination of 90 µmol photos m-2s-1at 25 °C. After reaching the exponential growth phase, the cyanobacterial strains will be harvested by centrifugation at 6,000×gfor 15 min at 4 °C, washed with DI water, and resuspended in P-free (or N-free) BG-11 medium. The strains will be cultured for 14 d to exhaust the stored nutrients (P or N) in algal cells without external nutrient provision.To ensure nutrient starvation, the OD of the cells will be monitored daily until no cell growth is observed and the ambient nutrient concentration is below the detection limit of the UV-vis spectrophotometer.After P (or N) starvation, each cyanobacterial strain will be inoculated in a 450 mL culturing jar containing 200 mL culture medium.The initial cell density of each strain will be adjusted to 1×106cells/mL, which is typical during our monthly monitoring of the aquaculture ponds. Different culture media containing the same TP (or TN) concentration will be prepared for the bioavailability tests using:1. >1,000 nm particles,2. 1,000-450 nm particles,3. <450-100 nm particles,4. <100-50 nm particles,5. <50-1 nm particles,6. the truly dissolved phase (<1 nm), and7. K2HPO4control (or KNO3control for N experiments).5. Test and use engineered biochars for removing nutrients from ponds.Lab-scale experiments will be systematically conducted to screen out the best combination of biochars (including ACs) for nutrient control and HAB mitigation. Lab sorption experiments for P and N in different size fractions and five algal parameters by different biochar and AC combinations will be tested in aqueous solutions with similar water chemistry to catfish aquaculture ponds.Once the best biochar combination (e.g., AC + eucalyptus 500 biochar + switchgrass 500 biochar) is obtained, a biochar vendor (e.g., Oregon Biochar Solutions who donated the commercial biochar) will be contacted for the mass production of biochars with similar quality. Wilson's lab has conducted whole pond scale experiments using engineered solutions (e.g., sorbents) for HAB mitigation in the aquaculture ponds in Alabama since catfish producers showed great interest in water quality improvement at pond scale. Depending on water quality data and the performance of the best biochar combination, the dosage of biochar for pond studies will be determined. Two mixing strategies for pond studies, including 1. fixed paddlewheel aerators and 2. mobile tractor paddlewheels, will be used to determine the effectiveness and efficiency of biochars for water quality improvement and HAB mitigation. Nutrients (P & N) and key algal parameters before and after biochar applications will be monitored to determine the effectiveness and efficiency of the engineered solutions (engineered biochars) for HAB mitigation in pond studies.

Progress 07/01/24 to 06/30/25

Outputs
Target Audience:The target audiences provided by the water quality monitoring project in catfish aquaculture ponds include: Catfish farmers: To improve their understanding of water quality monitoring and management and to adopt best practices for sustainable aquaculture. Fisheries and aquaculture researchers: To enhance their knowledge of water quality monitoring and management and to develop new research methods and technologies. Extension agents: To provide them with the knowledge and skills to effectively communicate and disseminate research findings to catfish farmers and other stakeholders. Policymakers: To educate them on the importance of water quality monitoring and management in catfish aquaculture and to inform policy decisions. Water quality professionals: To enhance their knowledge and skills in water quality monitoring and management, including sampling and analysis, modeling, and data interpretation. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provides ample, unique opportunities for training and professional development for the project team members and stakeholders, especially from the standpoint of nanoscience, nanotechnology, and water quality monitoring. Training and professional development opportunities include, but are not limited to the following: Field training: The foundation of this project is working directly at functioning catfish farms in the Blackbelt region of Alabama. During bimonthly sampling, project participants receive hands-on training where they collect water from 21 ponds spread across 5 different catfish production farms with a rigid plastic sampler. Project team members learn and practice water sampling techniques at the ponds with analytical tools to measure temperature, dissolved oxygen (DO), specific conductivity, and pH as well as a Secchi disk to measure water transparency. These data highlight seasonal shifts in ponds over time. Laboratory training: Project team members gain hands-on experience in laboratory procedures, especially with respect to the sequential particle size separation of pond water into large particles, colloids, nanoparticles, and truly dissolved phase. The new particle size separation approach involves various concepts related to water chemistry, particle size, particle density, Stokes' law, centrifugation, and nanoscience. Also, project team members learn skills and knowledge on the operation of various analytical instruments for nutrients and algal-related parameters, including UV-Vis spectrophotometer, gas chromatography-mass spectroscopy (GC-MS), and others at Auburn University and University of Florida. Hands-on experiences of using instruments for the characterization of nanomaterials: Project team members utilize various state-of-the-art instruments to characterize the colloids and nanomaterials separated from the pond water. Particle size, morphology, surface charge, surface functional group, elemental composition, and coupling of nutrients (P & N) and other major elements (e.g., Fe, Al, Ca) were characterized by using scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDS), zeta potential analyzer, particle size analyzer, etc. Water quality data analysis: Project team members learn how to use statistical tools to analyze water quality data to elucidate the relationship between nutrients on different particle size fractions with algal-related parameters. These data will enable us to narrow down sensitive water quality parameters for the forecasting of algal blooms. Best Management Practices (BMPs) for water quality management: Project team members learn BMPs for water quality monitoring and management in catfish aquaculture, such as proper waste management, water exchange, and aeration. Remediation strategies using engineered biochars will be a part of the BMPs that will be tested in Year 3 of this project. Extension and outreach: Project team members learn about research methods and extension avenues for water quality monitoring and management, including data collection and dissemination of research findings. More importantly, project team members learn about effective communication and outreach strategies to engage stakeholders, including catfish producers, policymakers, and the general public, on water quality issues and best practices. Collaboration and partnership building: Project team members learn about the importance of collaboration and partnership building among stakeholders, including researchers, catfish producers, policymakers, and industry representatives, to address water quality issues in catfish aquaculture. How have the results been disseminated to communities of interest?Project Director, Dr. Alan Wilson and his students collect samples at 21 catfish ponds spread across five farms in west Alabama (4 or 5 ponds at each farm). While sampling, he regularly talks with the catfish farmers about water quality challenges and successes. During these impromptu meetings, Dr. Wilson shares new research findings from his lab with the farmers but often leaves the meetings with more research questions to share with his students to develop new research projects to support the farmers. In October 2024, Project Director Wilson attended the Society of Environmental Toxicology and Chemistry in Ft. Worth, Texas, where he was a co-organizer of a session focused on algal blooms called "One Health of Planktonic, Pelagic and Benthic Harmful algal blooms (HABs): The detection, fate, effects, monitoring, and management of blooms". This session brought together a diverse set of speakers (students, academics, and agency scientists) to discuss current challenges and solutions for dealing with algal blooms. Wilson shared results from his catfish aquaculture pond monitoring efforts where he is using drones to monitor algal (and cyanobacterial) concentrations over time among 19 ponds. Two of Wilson's M.S. students also presented their research focused on controlling algal blooms in catfish production ponds. In December 2024, Project Director Wilson led an outreach event for two 9th grade science classes at Auburn Junior High School where he brought microscopes and plankton samples to excite the next generation of limnologists and water resource managers about the importance of microscopic aquatic organisms. In March 2025, Project Director Wilson attended the World Aquaculture Society Aquaculture America annual meeting in New Orleans where he chaired one session ("Mitigating Effects of Harmful Algal Blooms on Aquaculture"), filled in as chair for Dr. Tim Sullivan ("Impacts and Outcomes: USDA NIFA Support for U.S. Aquaculture"), gave an invited presentation titled "Harmful algal blooms in aquaculture: how to know when green water is bad" in the Aquatic Animal Health 101 session, and gave an oral presentation titled "Using unoccupied aerial systems to monitor algal blooms in catfish production ponds across seasons" in the Mitigating Effects of Harmful Algal Blooms on Aquaculture session. One of Wilson's M.S. students also presented their research focused on controlling algal blooms in catfish production ponds in the Mitigating Effects of Harmful Algal Blooms on Aquaculture session. In April 2025, Project Director Wilson and his colleague, Mr. Chuck Hemard (chair of the Auburn University Department of Art and Art History), were invited to talk with an Honor's College class about their efforts to integrate art and science. Wilson and Hemard have been photographing catfish ponds at one farm Co-Project Director, Dr. Dengjun Wang chaired three sessions, including "Monitoring, Prediction and Mitigation of Harmful Algal Blooms (#227431)" and "Advancements in the Fate, Transport, Transformation, and Remediation of Contaminants in the Environment (#223775)" at the America Geophysical Union (AGU) Fall Meeting 2024, and "Soil and Water Quality Impacted by Solute Transport and Remediation of Contaminants" at the 2024 ASA-CSSA-SSSA International Annual Meeting to widely disseminate the project findings to the scientific communities. Specifically, AGU Fall Meeting has an attendance of 25,000 - 30,000 and ASA-CSSA-SSSA also attracts thousands of researchers every year. Additionally, Dr. Wang was invited by USDA-NIFA program manager, Dr. Tim Sullivan to present this project at the Aquaculture 2025 (March 8, 2025) - a meeting directly geared towards aquaculture industry stakeholders, consumers, retailers, catfish industry investors, government agencies, policymakers, in addition to research institutions and academia. What do you plan to do during the next reporting period to accomplish the goals?The project is currently proceeding as planned. In Year 2, the project team plans to continue working on Objective 1: water sampling and Objective 2: nutrient and algal-related parameter analysis, as well as to start working on Objective 3 (bioavailability of nutrients on particles to cyanobacteria) and Objective 4 (nutrient control and HAB mitigation by engineered solutions). Furthermore, the project team will focus more on the delivery of project findings in scientific journals and at conferences. Especially, the project team will organize workshops and sessions to directly disseminate the project findings to direct stakeholders (e.g., catfish producers) and other stakeholders on water quality, HAB, best management practices, etc.

Impacts
What was accomplished under these goals? The year 1 activities of our team mainly focused on Objective 1 and Objective 2 of the project. Specifically, for Objective 1, our team conducted bimonthly water sampling of 21 catfish aquaculture ponds in west Alabama and employed the new particle size separation approach recently developed by our team (Hamid et al., 2023, Chemosphere, 340, 139906) to separate water samples into different size fractions. These include large particles, colloids, nanoparticles, and truly dissolved phases, so that we can accurately evaluate nutrient (e.g., P) load and bioavailability of these different particle size fractions in the pond water with respect to algal grow and harmful algal bloom (HAB) risk. With respect to Objective 2, our team analyzed nutrient concentration and speciation, as well as key algal-related water quality parameters (algal pigments, algal toxins, and off-flavor and odor compounds) of the pond water samples. The modified Hedley's extraction method was applied to characterize the speciation of P on different particle size fractions in pond water. Large datasets were obtained with the following key findings shown below: Nutrient (e.g., P) load, as reflected by particle concentration, in the 21 catfish aquaculture ponds exhibited geographical diversity and seasonal variation with growing season months (May-October) showing greater nutrient load and particle concentrations. Similarly, P concentration on different sized particles was higher in the summer months than in the winter months. Especially, pond water showed a higher presence of colloid- and nanoparticle-associated P, compared to other particle size fractions. Results from the modified Hedley's extraction method indicated that over 70% of both inorganic and organic P (Pi and Po) are accumulated in particles with sizes less than 50 nm. Notably, the nitric acid (HNO3)-extracted P--which is traditionally considered as the biologically unavailable form--was abundant and closely aligned with DI-water-extracted fractions. These results suggest that calcium phosphate minerals play an important role in P retention and release in aquaculture pond water. Statistical analyses based on multi-correlation analyses indicated that soluble reactive P (SRP) concentrations are positively correlated to water hardness and negatively correlated to dissolved oxygen (DO) content, underscoring the influence of redox conditions and ionic interactions on P mobility. Our findings also emphasize the overlooked role of detrital P, particularly calcium-bound nanoparticle P, in aquaculture ponds and highlight the need to reassess its contribution to harmful algal blooms (HABs) in aquaculture ponds.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Le, V. V. and A. E. Wilson. In press. A handheld fluorometer evaluates freshwater cyanobacterial blooms across a broad productivity gradient. Lake and Reservoir Management
• Type: Other Journal Articles Status: Published Year Published: 2024 Citation: Wilson, A. E., H. Zinnert, S. S. Ganegoda, P. P. Johnson, D. Wang, H. A. Torbert, and B. H. Beck. 2024. Gypsum increases soluble reactive phosphorus and blue-green algae in catfish production ponds. Alabama Fish Farming Center 2:12-13.
• Type: Other Journal Articles Status: Published Year Published: 2024 Citation: Wang, D., A. K. Hamid, I. M. Radwan, A. E. Wilson, H. A. Torbert, and B. H. Beck. 2024. Understanding how phosphorus could be removed in aquaculture ponds by gypsum. Alabama Fish Farming Center 1:12-13.