Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 133 - Pollution Prevention and Mitigation | 5220 - Pesticides (includes herbicides, insecticides, fungicides, etc.) | 1010 - Nutrition and metabolism | 100% |
Large-scale livestock farms with concentrated animal feeding operations (CAFOs) have replaced traditional household breeding to meet the increasing demand for animal products. However, this transformation has generated a significant volume of livestock wastewater with a large amount of nutrients and antibiotics. There is solid evidence of the significant contribution of livestock farms to the impairment of surface water and groundwater. However, no studies propose an effective and sustainable management strategy for their remediation from livestock wastewaters. Intensive livestock farming generates huge quantities of livestock wastewater with high concentrations of nutrients and antibiotics, which leads to the deterioration of surrounding freshwater ecosystems without appropriate management. On the other hand, using the nutrients in wastewater to cultivate microalgae is a promising strategy with the advantages of removing nutrients and micropollutants from wastewater and bringing additional values by using the harvested microalgae as a feedstock for biofuel production. There is an urgent need to identify the best management strategies for reducing loads of nutrients and antibiotics from livestock wastewater to freshwater ecosystems via microalgae cultivation and develop the microalgae process models to optimize the pond operations and management practices.Our project proposes identifying effective microalgae strains using livestock wastewater as a growth medium for integrating the removal of multiple nutrients and antibiotics.We will optimize initial nutrient compositions and management practices in microalgae cultivation and harvesting for effective nutrient and antibiotic reduction from livestock wastewater before discharge to surface water or groundwater. Moreover, we will conduct field experiments to cultivate microalgae in open storage ponds in farms and develop microalgae models to predict microalgae growth and nutrient removal and optimize pond operations and management practices.Both food productivity and healthy water quality at local, regional, national, and global scales are critical to sustainable agriculture. This project addresses enhancement of agricultural and natural resource sustainability by combining one of the fastest growing plants - microalgae with animal production practices of CAFOs for the long-term goals: - 1) help meet human food need on animal products; 2) enhance environmental quality and a natural resource base (water) necessary for agriculture; 3) make efficient use of on-farm resources (retaining ponds and lagoons) by integrating natural biological cycles (nutrient cycles) and controls (microalgae), which can, in turn, 4) sustain the economic viability of farm operations through the production of microalgae biomass that can be harvested for bioenergy production. The work represents the first integrated attempt to remediate the nutrients and antibiotics in livestock wastewater from a circular bioeconomy perspective. Thus, understanding these processes and interactions and finding practical and economic mitigation approaches would help maintain the highest food crop productivity without compromising water quality in natural environments.
The improper management of livestock wastewater has deteriorated freshwater ecosystems due to the heavy loads of nutrients and antibiotics. Large-scale livestock farms with concentrated animal feeding operations have replaced traditional household breeding to meet the increasing demand for animal products. However, this transformation has generated a large volume of livestock wastewater with high nutrient and antibiotic concentrations. It is a particular concern on reducing loads of nutrients and antibiotics from livestock wastewater to surrounding water bodies. Using wastewater to cultivate microalgae has recently attracted broad attention due to its advantages of using nutrients for microalgae growth, removing micropollutants, and bringing additional values via using microalgae for biofuel production. To identify the best management practices for nutrient and antibiotic removals, we propose a four-year study with three specific objectives:
With support from the Piedmont Research and Education Center at Clemson University, we will use livestock wastewater to cultivate microalgae in three storage ponds for over one year. We will use the laboratory and field data to develop the process models to predict microalgae growth and nutrient removals and optimize pond operations and management practices for maximum nutrient removals. Additionally, we will use microalgae for biofuel production and conduct the techno-economic and life cycle analyses.
i) General Water Quality and Nutrients (Clemson University): Water samples will be filtered through 0.45 membrane filters and analyzed for nutrients including total organic carbon (TOC) and total nitrogen (TN) using a TOC/TN analyzer (Shimadzu, Japan) and inorganic ions of NH4+, NO2-, NO3-, and PO43- using a Dionex ICS-1500 ion chromatography system (Thermo Scientific, US) (Majidzadeh et al., 2017 *). Total phosphate (TP) will be determined using the ascorbic acid method following US EPA 365.3. General water quality, including temperature (°C), EC (mS/m), pH, and DO (mg/L) will be measured by a YSI multi-parameter electrode.ii) Analysis of Antibiotics (Clemson University and USDA-ARS): Water samples will be filtered through two pieces of glass microfiber filters (0.7-μm and 2.7-μm, Whatman Inc, NJ). The cleanup procedure will be based on the method from Vanderford and Snyder (2006). The target compounds will be extracted from aqueous samples onto Oasis hydrophilic-lipophilic balance (HLB) solid-phase extraction (SPE) cartridges. Cartridges will be sequentially preconditioned with methyl tert-butyl ether (MTBE), methanol, and reagent water. After the conditioning step, 1 L of samples will then be loaded onto the cartridges at 10 mL/min, after which the cartridges will be rinsed with 5 mL of reagent water and then dried under vacuum for 30 min. The SPE cartridges will be eluted with 5 mL of methanol followed by 5 mL of 10/90 (v/v) methanol/MTBE. The eluents will be evaporated to less than 0.5 mL using a Rapid Vap system (Model 79000-02, Labconco Co., MO) and brought up to a final volume of 0.5 mL using methanol before analysis with LC-MS/MS. Antibiotics will be extracted from the collected solid samples (including grinded microalgae) using an ultrasonic cleaner (Mettler Electronics Corp, CA). The extraction procedure followed modified methods by Wu et al. (2011) and Vanderford and Snyder (2006). Before extraction, filtered water samples and solid samples will be spiked with a solution of isotopically labeled standards that contain a stable isotope of analyte (sulfamethoxazole-d4, tetracycline-d6, ciprofloxacin-d8) to assess compound recovery and accuracy. The details of analytical methods, instruments, and analyses are provided in Yang et al. (2016) *. Additionally, the collected samples can be analyzed at L.G. Rich Environmental Research Laboratory at Clemson University using Agilent 1290 LC with Agilent 6470 triple quadruple mass spectrometer (Agilent, US) after the online pre-concentration by an automated Agilent 1290 Infinity II Flexible Cube.iii) Lipids/Biodiesel (Clemson University): Total fatty acids composition in microalgae lipids will be determined by gas chromatograph (Shimazu GC-2010) equipped with a flame ionization detector (FID) with an Rt-2560 column (100 m × 0.25 mm × 0.2 µm) and helium as carrier gas. We will use Supelco 37 component FAME mix from Sigma as FAMEs standard and pentadecane (100 µg/mL) and C13:0 (200 µg/mL) as internal standard. Total fatty acid concentration will be calculated by summing the concentrations of each fatty acid component together. Total carbohydrates will be determined using the phenol-sulfuric acid method with glucose as the standard. Total protein will be determined using the modified Lowry protein assay kit (Thermo Scientific, US). The carbohydrate composition of the hydrolysate will be determined by HPLC with a Shodex Sugar SP0810 (7 µm, 8 × 300 mm) column and a Shodex RI (101) detector using the mobile phase of water at 0.6 mL/min and 80 °C. Biodiesel composition will be measured by GC-FID with a SP-2560 capillary column.iv) Bacterial and Microalgae DNA (Clemson University): Samples at the mid-exponential phase of microalgae growth will be collected in sterile containers and quantified following the method in Udaiyappan et al., (2020). Specifically, we will use the Wizard® Genomic DNA Purification (Promega, USA) kit following the manufacturer's protocol to extract bacterial and microalgae DNA, which will then be stored at -20 ? before analysis. We will determine DNA concentration using a Qubit® Fluorometer (Invitrogen, Qubit-IT™ dsDNA HS Assay kit) and perform the Quantitative real-time polymerase chain reaction (qRT-PCR) using the StepOne Real Time PCR System (Applied Biosystems) at the L.G. Rich Environmental Research Laboratory at Clemson University. Illumina MiSeq genomic analysis on purified DNA amplicons will be performed at Clemson University Genomics and Bioinformatics Facility (CUGBF). Illumina MiSeq data from CUGBF will be analyzed using QIIME to determine the presence of specific 16S rDNA sequences.v) Techno-Economic and Life Cycle Analyses (Clemson University): We will use SuperPro Designer software (intelligen.com) to perform a techno-economic analysis by including the process of cultivation, harvesting, oil extraction, degumming, and biodiesel production. We will calculate the mass and energy balances for each unit operation in the process. The simulation parameters and stoichiometric equations will be formulated using the data from Tetramer Technologies. The outcomes will include capital cost (direct fixed capital and indirect capital cost), operating expenditure (raw materials, labor, consumables, laboratory test, waste disposal, utilities, miscellaneous cost, etc.), and profitability (gross margin, return of investment, payback time). We will also conduct the life cycle assessment analysis using the LCA software and databases at Clemson University.
Target Audience
Scientists and engineers who are passionate to know a better and more effective nutrient recycling practice to reduce nutrients and antibiotics transfer into freshwater ecosystems. Moreover, environmentalists who are striving to devise risk reduction and mitigation strategies might be interested. Local agencies, farmers, landowners, and agriculturists who manage large-scale livestock farms using concentrated animal feeding operations (CAFOs).
Changes / Problems
Dr. Chow, one of the co-project directors at Clemson University, left the university. Dr. Walker, one of the co-project directors at Clemson University,retired in 2024 summer.
Training & Professional Development
This grant so far supports to train three undergraduate students and two graduate students at Clemson University Yu-Hua Zheng - graduate student in Forest Resources at Clemson University Pei-Lin Tan - graduate student in Forest Resources at Clemson University Austin Allen - undergraduate student in Biosystem Engineering at Clemson University Anthony Villanueva - undergraduate student in Biosystem Engineering at Clemson University Erika Carter - undergraduate student in Biosystem Engineering at Clemson University
Dissemination Streams
We have presented the data from this project at three conferences. We are preparing one manuscript based on the data we obtained from the first and second years, which will be submitted to a peer-reviewed journal soon.
Next Reporting Steps
Plan Overview We plan to conduct experiments to investigate the controlling factors on nutrient recovery and potential biodiesel production. Objective I - Microalgae Strains on Nutrient and Antibiotic Removal This has been completed. Objective II - Controlling Factors for Nutrient and Antibiotic Removal We are conducting experiments such as adding additives to improve the microalgae growth when using undiluted dairy LWW. ?Objective III - Process Models for Pond Operations Once we accomplish objectives I and II, according to the experimental findings and observations, we will optimize the process of microalgae cultivation for reducing the nutrient and antibiotic loads to surrounding water bodies, improve the process-based models to optimize pond operations and management practices for the nutrient removal, and examine the economic feasibility of using harvested microalgae as a feedstock for biofuel production.
Target Audience
1. Scientists and engineers who are passionate to know a better and more effective nutrient recycling practice to reduce nutrients and antibiotics transfer into freshwater ecosystems. Moreover, environmentalists who are striving to devise risk reduction and mitigation strategies might be interested. 2. Local agencies, farmers, landowners, and agriculturists who manage large-scale livestock farms using concentrated animal feeding operations (CAFOs).
Changes / Problems
Dr. Rui Xiao, one of the co-project directors at Clemson University, left the university.
Training & Professional Development
This grant so far supports to train one graduate and three undergraduate students at Clemson University Pei-Lin Tan - graduate student in Forest Resources at Clemson University Austin Allen - undergraduate student in Biosystem Engineering at Clemson University Anthony Villanueva - undergraduate student in Biosystem Engineering at Clemson University Erika Carter - undergraduate student in Biosystem Engineering at Clemson University
Dissemination Streams
Nothing Reported
Next Reporting Steps
Plan Overview We will complete the objective of identifying microalgae strains for nutrient and antibiotic removal. We plan to conduct electrocoagulation experiments to investigate the cost-effective approach for microalgae harvesting. Objective I - Microalgae Strains on Nutrient and Antibiotic Removal We have already identified two microalgae species that can grow by using the nutrients from dairy livestock wastewater. We will continue studying the influence of growing conditions on microalgae growth. We will proceed with evaluating the influence of rainwater dilution factor, antibiotic concentrations, NaHCO3 concentrations, etc. We expect that this objective of selecting microalgae strains using livestock wastewater as a growth medium for integrating microalgae biomass production and nutrient and antibiotic removal will be completed by the next reporting period. Objective II - Controlling Factors for Nutrient and Antibiotic Removal Once we accomplish objective I, we will start the proposed experiment in this objective to understand the controlling factors for nutrient and antibiotic removal. Objective III - Process Models for Pond Operations Once we accomplish objectives I and II, according to the experimental findings and observations, we will optimize the process of microalgae cultivation for reducing the nutrient and antibiotic loads to surrounding water bodies, improve the process-based models to optimize pond operations and management practices for the nutrient removal, and examine the economic feasibility of using harvested microalgae as a feedstock for biofuel production. <br><br>
<br>What was accomplished under these goals? Progress Overview: For the first year of the project at Clemson University (CU), we worked on how to select microalgae strains using livestock wastewater as a growth medium for integrating microalgae biomass production and nutrient and antibiotic removal as proposed (Objective I). We organized a symposium entitled "Occurrence, Transport, and Fate of Contaminants of Emerging Concern (CECs) in Agroecosystems" during the Spring 2023 ACS Meeting. We are using the data we obtained from the microalgae experiment in the first year to submit an oral presentation to the 2nd International Online Conference on Agriculture (1-15 Nov 2023). Objective I - Microalgae Strains on Nutrient and Antibiotic Removal In our studies at Clemson University, we collected the dairy livestock water from a large water lagoon in the Lamaster Dairy Farm (~200 heads; Anderson, SC) in Piedmont Research and Education Center (REC) on June 24, 2022. This pond is used for storing livestock wastewater before reuse for irrigation or flushing the freestall barn. The collected dairy livestock water was filtered through Whatman Glass Microfiber filters (CAT No.: 1827-047) and then used to cultivate two microalgae species of Chlamydomona reinhardtii and Chlorella protothecoides (purchased from UTEX Culture Collection of Algae at UT-Austin) from 2022 July. Specifically, on July 5 2022, we added 100 mL of distilled and deionized water (DDI water) and 1 mL of BG-11 into a sterile 500 mL Erlenmeyer flask to prepare the liquid medium. The 10 µL inoculation loop was used to transfer a noticeable amount of microalgae culture into the flask with the liquid medium, which was then placed in a shaker (Cat# 89032-092; VWR orbital shaker) under the simulated sunlight (2000-3000 LUX) for cultivation (24 h light on). On August 3, 2022, to prepare microalgae in 25% livestock wastewater, we added 20 mL of microalgae solution from the 500 mL Erlenmeyer flask (July 5; green color), 25 mL of filtered livestock wastewater, and 55 mL of the liquid medium (containing BG-11) into a new 500 mL Erlenmeyer flask, which was then placed in a shaker under the simulated sunlight (2000-3000 LUX) for cultivation (24 h light on). Three replicates were conducted for each microalgae species. On September 13, 2022, to prepare microalgae in 50% livestock wastewater, we added 20 mL of microalgae solution from the 500 mL Erlenmeyer flask (August 3; green color), 50 mL of filtered livestock wastewater, and 30 mL of DDI water into a new 500 mL Erlenmeyer flask, which was then placed in a shaker under the simulated sunlight (2000-3000 LUX) for cultivation (24 h light on). Three replicates were conducted for each microalgae species. On October 11, 2022, to prepare microalgae in 75% livestock wastewater, we added 25 mL of microalgae solution from the 500 mL Erlenmeyer flask (September 13; green color) and 75 mL of filtered livestock wastewater into a new 500 mL Erlenmeyer flask, which was then placed in a shaker under the simulated sunlight (2000-3000 LUX) for cultivation (24 h light on). Three replicates were conducted for each microalgae species. On December 14, 2022, to prepare microalgae in 100% livestock wastewater, we added 20 mL of microalgae solution from the 500 mL Erlenmeyer flask (October 11; green color) and 80 mL of filtered livestock wastewater into a new 500 mL Erlenmeyer flask, which was then placed in a shaker under the simulated sunlight (2000-3000 LUX) for cultivation (24 h light on). Three replicates were conducted for each microalgae species. We testified the influence of simulated sunlight intensity (1000 LUX, 3000 LUX, 5000 LUX, and 7000 LUX) on the microalgae growth and the nutrient recovery. Specifically, we centrifuged 10 mL of the cultivated microalgae at the stationary phase, which was transferred into the 500 mL Erlenmeyer flask with 100 mL of filtered livestock wastewater after dumping the supernatant. The Erlenmeyer flask was placed in a shaker under the simulated sunlight for cultivation (12 h light on and 12 h light off; VIPARSPECTRA Led Grow Lights) inside a grow tent (VIVOSUN). The sunlight intensity was measured regularly at five plots on the shaker using BT-881D digital illuminance light meter. We measured OD680 regularly to monitor the microalgae growth and the experiments were stopped when the stationary phase was reached. When the experiments were stopped, the microalgae solution inside the 500 mL Erlenmeyer flask was centrifuged - the supernatant was stored for measuring the water quality including nutrients and the microalgae sediment was freezing dried for measuring the total fatty acids. Three replicates were conducted for each microalgae species under each sunlight intensity. Objective II - Controlling Factors for Nutrient and Antibiotic Removal After we complete the tasks proposed in Objective I, we will move on to evaluate the influence of the biochemical compositions of livestock wastewaters on nutrient and antibiotic removal. We will determine the controlling factors in microalgae cultivation on nutrient and antibiotic removal. Besides, we will assess the controlling factors in microalgae harvesting on harvesting efficiency along with nutrient and antibiotic removal. Objective III - Process Models for Pond Operations No work on this objective is done in the first year. <br><br><b>Publications</b><br>