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%
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.