Research Project: Developing Agronomically and Environmentally Beneficial Management Practices to Increase the Sustainability and Safety of Animal Manure Utilization

Location: Food Animal Environmental Systems Research

2023 Annual Report

Objectives
Obj 1: Improve utilization of animal manure and manure byproducts, such as biochar and hydrochar, to increase soil health and agricultural productivity, and protect the environment. 1.1 To evaluate the use of biochars and hydrochars for reducing phosphorus release from high P soils. 1.2 Application of biochar over the bedding materials inside the poultry house to reduce ammonia emission and improving the quality of the resulting poultry litter for crop production. 1.3 Evaluate biochar effects on composting cattle manure to mitigate carryover effects of pyridine herbicide. 1.4 Develop a sustainable management practice using poultry litter and cover crop for maximum nutrient use efficiency under a corn production system. Obj 2: Quantify, characterize, and develop strategies to reduce atmospheric emissions, including gases, particulate matter, and other emissions of concern, from animal production facilities and manure application sites. 2.1 Evaluate the fate of atmospheric emissions from agricultural systems. 2.2 Characterize the spatial and temporal profiles of nitrogen in the gas and particle phase from agricultural emissions. 2.3 Model the physical processes that are responsible for the production of atmospheric aerosols from farm waste compounds. 2.4 Reduce malodors characteristic of animal wastes by the use of high sugar containing agricultural by-products. 2.5 Develop novel and cost-effective nano-filtration materials to capture ammonia, GHG, and odors from livestock production facilities. Obj 3: Develop mathematical models and evaluate practices for managing and treating manure to obtain clean bioenergy; reduce gaseous emissions; minimize losses of nutrients, estrogens, hormones, and other compounds of concern; and control the survival and transport of antibiotics, antimicrobial resistant bacteria, and foodborne pathogens. 3.1 Evaluation and optimization of naturally occurring enzymes to improve anaerobic digestion of recalcitrant livestock wastes to reduce emissions and obtain bioenergy for sustainability. 3.2 Use of sound to enhance biogas production and reduce sludge from animal production facilities. 3.3 Investigate effects of supplemental ionophores on animal performance, manure nutrient composition, and soil chemical and biological characteristics of cattle grazing tall fescue or cool-season annuals. 3.4 Quantifying estrogens and their sulfonated conjugates originating from dairy practices. 3.5 Develop, calibrate, and evaluate field-scale fate and transport models for describing P loss from agricultural fields in the Western Lake Erie Basin (WLEB). 3.6 Modeling transport, fate, and risk management of nitrate and common and emerging pathogens in agriculturally-impacted groundwater systems. 3.7 Characterize the epidemiology, serotype distribution and antimicrobial resistance of Salmonella in broiler production system. 3.8 Determine the prevalence and resistance genes of extended spectrum beta-lactamase (ESBLs) producing Escherichia coli in broiler production system.

Approach
A significant challenge facing U.S. agriculture is how to safely and sustainably manage the more than one billion tons of animal manure produced annually. Our research is directed at optimizing methods of manure application, storage, and treatment to improve agricultural productivity while protecting the environment and human health. Our research will investigate the use of poultry litter for improving crop yield and soil health. We will evaluate novel methods for using biochar to reduce phosphorus release from soils to protect water quality, to reduce ammonia levels in poultry houses, and to reduce the release of pesticides from composted manure. To protect air quality we will characterize and identify formation processes of ammonia, amine, and aerosol emissions from agricultural facilities; develop treatment technologies for reducing emissions from livestock production facilities; develop cost-effective nano-filtration materials to capture ammonia, greenhouse gases, and odors from livestock production facilities; and evaluate the use of acoustic technology to reduce gas emissions from anaerobic digestors. We will identify enzymes to optimize biogas production from recalcitrant livestock wastes, test whether manure nutrient composition can be modified by feeding ionospheres to cattle, and study the fate of estrogens following anaerobic digestion of dairy cattle waste. Our research will test the accuracy of field-scale models for predicting phosphorus loss from tile-drained agricultural fields. We will investigate the transport of bacteria in karst topography. Finally, our research will help protect human health by defining the epidemiology and antimicrobial susceptibility of major Salmonella serotypes circulating in broiler production systems and the prevalence of extended spectrum beta-lactamase producing E. coli in broiler production. Results will be disseminated to the public through fact sheets, web-accessible models, stakeholder meetings, and publications in scientific journals and will benefit the general public by providing much needed information for developing more effective strategies for sustainably managing animal manure.

Progress Report
In support of Sub-objective 1.1, experiments were initiated to measure the kinetic sorption of phosphorus (P) to four soils amended with untreated and iron-coated biochar. We have found that the sorption of P to both biochars reaches equilibrium in approximately 4 hours whereas it takes several weeks to reach equilibrium in the four soils. When the biochar was added to the soils, the amount of P sorption increased for each soil with the iron-coated biochar but the overall kinetic behavior did not change. The effect of the iron-coated biochar was most pronounced in a sandy soil and least pronounced in a silt-loam soil. Addition of the uncoated biochar did not affect P sorption to any of the four soils. In support of Sub-objective 1.2, we acquired hardwood biochar and entirely characterized its constituents. A local poultry producer agreed to collaborate with us and allowed us to use one of his commercial-size poultry houses for this study (experiment A). The house was instrumented before biochar application to half of the poultry house. The ammonia gas (NH3) concentration in the house was measured continuously until the flock was harvested. Integrated biochar and poultry litter was collected after the flock was harvested. Preparation is ongoing to conduct the field plot study as part of experiment B. In support of Sub-objective 1.3, biochar was applied at different rates (0, 2, 4 and 10%) to compost containing the herbicide aminopyralid and a bioassay test was performed with the compost using tomato seedlings, to determine if the biochar would bind the herbicide. Results showed that treatments with 4% and 10% biochar levels did not contain any residual aminopyralid concentrations and mixing compost start mixture with 4% and 10% biochar levels can reduce residual effect of herbicides on crops grown on compost material. However, the treatments with 4% and 10% biochar showed mild symptoms in bioassay tests with lower injury index values. In support of Sub-objective 1.4, a field plot study was established to investigate soil health management on plant nutrient uptake, along with the overall mass balance of nitrogen (N). This field study will determine the fate of the applied poultry litter and chemical N in a continuous corn rotation. We collected background soil samples, applied treatments, planted first-year crops, performed soil chemical analysis, and installed chambers for greenhouse gas (GHG) measurements. We applied second-year treatments to the same plots, took yearly soil samples prior to planting, and continued GHG monitoring. In support of Sub-objective 2.1A, environmental chamber experiments for waste emissions from swine, dairy, and poultry have been conducted. More experiments are in progress to study the variability of different waste parameters on air emissions and chemistry. In support of Sub-objective 2.1B, atomistic computational models have been made that adequately simulate the process by which sulfur dioxide (SO2) interacts with an air-water interface. The latest simulations show that the SO2 molecule prefers the surface and interior of a water droplet over being in the gas phase, regardless of speed or angle of impact of the initial collision, though the time at the surface depends on the concentration of ions in the solution. Work on the ionic strength effects is ongoing, as are comparisons to other molecules present in the atmosphere. In support of Sub-objective 2.2, passive air samplers have been deployed to several locations including poultry and dairy houses and have successfully given ammonia measurements and a procedure ready for the ARS ADAPT (Ammonia Deposition Project Team) study in Bowling Green, Kentucky. Full implementation of an ADAPT site focused on a poultry house location is anticipated in year three. Our research indicates that the sampling protocol that the ADAPT project will be using for ammonia will not be adequate for the organic nitrogen compounds. We are developing a denuder method that can be co-located with the passive air samplers at the ADAPT sites, both in Bowling Green, Kentucky, and elsewhere, so that organic nitrogen information will still be obtained during the project as anticipated in the project plan. In support of Sub-objective 2.3, research continued on refining and optimizing our process for modeling the chemical processes involved in the formation of SO2 from farm waste compounds (particularly reduced sulfur species). We have refined and automated several of the processes involved in identifying chemical pathways, in preparation for the next round of data expected from our Riverside, California, collaborators. In support of Sub-objective 2.4, dairy manure was treated with the sugars: sucrose, lactose, and glucose to determine if the treatment could reduce the concentrations of compounds such as p-cresol, p-ethylphenol, and skatole which are responsible for manure malodors. Instead of producing these compounds, sugar-treated wastewater produced fruit-like esters such as butyl acetate and butyl butyrate. In support of Sub-objective 2.5, the optimization of nanoparticles formation from plant extracts was studied to examine their feasibility in air pollution reduction, antimicrobial property and delivery of enzyme treatment. In addition, pilot-scale sequential filtration-scrubber system to utilize nanoparticles and nanofibers from agricultural residues for agricultural air pollution mitigation is in progress. In support of Sub-objective 3.1, we are continuing to monitor and optimize methane production from an anaerobic digestion system (15 million gallons) of poultry litter mixed with food wastes and evaluation of different anaerobic digestion systems on biogas production from recalcitrant livestock waste feedstocks. In support of Sub-objective 3.2, pilot scale anaerobic digesters were rebuilt inside a building dedicated to the digesters, heating and electricity were installed in the building, and ground condensers were installed on gas lines to remove moisture from the biogas and new sound systems were installed in the digesters. Post-digestion tanks were installed that will be used to remove phosphorus and nitrogen from the wastewater. In support of Sub-objective 3.3, grazing research using weaned cattle was conducted. Cattle grazed for 34 days on either tall fescue or annual ryegrass. Cattle were weighed at the beginning and end of the grazing period and manure, forage, and soil samples were collected every 14 days. Data from the study indicated an effect of forage type on cattle weight gain (0.5 pounds of additional daily body weight gain from grazing annual ryegrass compared with tall fescue), while data from manure and soil samples are still being analyzed by a commercial laboratory. In support of Sub-objective 3.4, research developing and testing a new analytical method to measure urine estrone and Beta-estriol concentrations over the estrous cycle of six dairy cows was completed. This method allows for determining free and conjugated forms of estrone and Beta-estriol in this sample type and can reach parts per trillion detection limits. In support of Sub-objective 3.5, research continued developing a runoff model that can adequately predict runoff from tile-drained fields. We have extracted all rainfall and runoff events, matched runoff events to rainfall events, and are currently applying different models to the data. Once completed, this runoff component will be incorporated into the Annual Phosphorus Loss Estimator (APLE) model to predict phosphorus losses from tile-drained fields in the Western Lake Erie Basin. In support of Sub-objective 3.6, research continued analyzing over 700 groundwater samples to identify and measure the type and quantity of bacteria, with a focus on antibiotic resistant bacteria (ARB) strains, including E. coli and other common resistance types. Preliminary Spring field sampling occurred at two test sites to determine potential resolution and water quality parameter monitoring success for sourcing and sampling protocols for high-resolution, focused data collection for E. coli, ARB, and nitrates during the prep and growing seasons. Fieldwork preparation to complete instrument installation for high-resolution monitoring, including a new optical nitrate probe system, is underway to collect E. coli, ARB, and nitrate data. In support of Sub-objectives 3.7 & 3.8, a field experiment was conducted to evaluate biochar poultry litter amendment as a control measure for Salmonella and extended spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) in broiler production. Biochar was surface applied to the back half of a broiler house at a commercial broiler production farm. Poultry litter samples were collected on five occasions: one day after biochar was applied and one day prior to chicken were introduced, thereafter approximately every two weeks during the full flock cycle. Samples were also collected from another poultry house with no biochar. Samples were cultured for Salmonella and ESBL-producing E. coli. Samples are being processed for DNA extraction and whole genome sequencing.

Accomplishments
1. Two-stage anaerobic digestion systems reduce unwanted agricultural wastes and improve bioenergy production for on-farm usage. Population growth has resulted in a greater demand for food, energy, and a greater generation of waste. Much of this waste is derived from agricultural activity and sent directly into landfills or incinerated with other wastes. Anaerobic digestion of these wastes may be a good option for agricultural residues disposal and could supply energy demands on-farm. However, the potential of biogas production from plant woody materials waste is not well recognized due to the low yields obtained. ARS scientists from Bowling Green, Kentucky, in collaboration with researchers in Mexico, evaluated two-stage (hydrolysis/acedogenesis vs. methanogenesis) anaerobic digestion systems using plant silage as feedstock. The results show that a higher degradation efficiency and energy conversion occurred from the two-stage anaerobic digestion system. In addition, two biofuels (hydrogen and methane) with high energy value can be obtained continuously in separate reactors. Therefore, the implementation of this two-stage anaerobic digestion technology could reduce unwanted agricultural organic wastes as well as obtaining renewable energy for on-farm utilization, resulting in benefits to agricultural production.

2. Multidrug resistant and human-disease causing Salmonella detected in karst groundwater and a groundwater fed surface water site. Nontyphoidal Salmonella causes an estimated 1 million illnesses, 20,000 hospitalizations, 400 deaths, and $400 million in direct medical costs per year in the United States. An estimated 212,500 infections and 70 deaths are attributed to drug resistant strains each year. Salmonella infections in humans are primarily foodborne, but a quarter of cases can be acquired from other sources including water. Karst groundwater is a major source of drinking water around the world; however, it is prone to bacterial contaminants such as Salmonella from the surface. Researchers from ARS in Bowling Green, Kentucky, Western Kentucky University, and Food and Drug Administration longitudinally monitored Salmonella prevalence in nine karst groundwater and one groundwater-fed river sites in Bowling Green by weekly sampling for 46 weeks. Salmonella was detected from 14.5% of samples, and from all sampled sites. The highest prevalence was detected in the river site, and during summer season. Salmonella isolates belonged to 18 serotypes, nine of which were among the top 20 serotypes commonly associated with human infections. Based on genome analysis, over two-thirds of the isolates did not match any genome in the national database; the remaining one-third matched Salmonella genomes of human or food animal origin, suggesting the importance of humans and animals as a source of groundwater contamination. Drug resistance was detected in 12.5% of the isolates, and most were multidrug resistant. The study revealed the occurrence of Salmonella including multidrug resistant, and strains commonly associated with human infections in ground and surface waters indicating the importance of surveillance and water treatment to alleviate a potential public health risk.

3. Cow-calf production is a reservoir for antibiotic-resistant bacteria of public health importance. Cow-calf operations are a large segment of the U.S. beef production system. Weaned calves are brought into feedlots to be finished for meat, and culled cows are slaughtered for beef, primarily for ground beef products. Stocking density and overall antibiotic use are low in these systems because both cows and calves are raised on pastures until weaning. Previous studies showed that the levels of antibiotic resistant bacteria in feedlot cattle, where both the stocking density and antibiotic use are high, gradually increase during the feeding period. However, studies in cow-calf operations are rare. Low level bacterial prevalence, including the antibiotic resistant population, can be carried over to the feedlot, persist, and propagate during the feedlot cycle. Understanding the baseline levels of bacteria under cow-calf production would contribute to develop preharvest beef safety mitigation strategies. ARS and Western Kentucky University researchers in Bowling Green, Kentucky, and ARS researchers in Clay Center, Nebraska, studied dynamics of bacteria resistant to important antibiotics for human health in the cow-calf production system. Weekly fecal samples both from the cows and calves were collected for 2-3 weeks before weaning. Overall prevalence of bacterial resistance was 97% for tetracycline resistant enterococci, 87% for tetracycline resistant Escherichia coli, 43% for erythromycin resistant enterococci, 31% for third generation cephalosporin resistant Escherichia coli, and 3% for extended spectrum beta-lactamase enzyme producing Escherichia coli. This study showed that bacteria resistant to antibiotics of importance of public health in cow-calf production where disease burden and antibiotic uses are low. It is essential to follow pre-weaned calves into the feedlot to understand if the levels of antibiotic resistant bacteria increase during the feedlot cycle.

4. Tylosin use in feedlot cattle increases foodborne macrolide resistant enterococci that can directly infect humans or transfer their resistance genes to other human pathogens. Liver abscess causes an estimated $16 million in economic loss annually to the beef cattle industry through liver condemnation, reduced animal performance, and carcass yield. Continuous use of tylosin antibiotic in the ration of feedlot cattle is the most effective approach to prevent liver abscess, but the collateral effect of tylosin use on the level of antimicrobial resistant bacteria needs more studies. Tylosin is animal only antibiotic but it belongs to macrolide class of antimicrobials that also includes erythromycin. Erythromycin is essential for the treatment of severe bacterial infections in humans. Since tylosin is analogous to erythromycin, its use in feedlot cattle can select for erythromycin resistant foodborne bacteria such as enterococci. ARS and Western Kentucky University researchers in Bowling Green, Kentucky, evaluated the effect of in-feed use of tylosin in feedlot cattle on erythromycin resistant enterococci bacteria and concentrations of tylosin in the animal feces. Higher macrolide resistant enterococci and tylosin antibiotic were observed in the feces of tylosin treated animals. Risk-based approaches including label changes to limit tylosin use, and development of effective manure treatments, are potential areas of research to reduce environmental and public health impacts resulting from tylosin use in U.S. beef cattle industry.

5. Development of a spreadsheet for determining critical soil test values, which can be used to ensure the appropriate application of phosphorus fertilizer to fields. Inorganic fertilizer is often applied to fields to maximize crop yields. Soil tests are often used to determine how much fertilizer to add to the fields. Researchers at ARS in Bowling Green, Kentucky, in collaboration with researchers at Kansas State University, North Carolina State University, Pennsylvania State University, and the University of Arkansas developed a spreadsheet for determining the relationship between soil test and crop response data to identify a critical soil test value (CSTV). The use of the spreadsheet will help state agencies update their fertilizer recommendations, which will lead to more efficient fertilizer use by identifying fields that require fertilizer to maximize yields while also identifying fields that are unlikely responsive to additional fertilizer inputs thereby reducing costs and protecting the environment.

6. Bird activity is associated with the release of particulate matter and odor from broiler houses potentially impacting animal and human health. Particle size and chemical composition of particulate matter may contribute to health effects and the odor associated with broiler houses. A scientist from ARS in Bowling Green, Kentucky, and researchers at Western Kentucky University, and Claremont Colleges measured particulate size distributions and ionic composition, and some gas-phase precursors at a broiler house to see if they were influenced by specific management practices. Particulate matter and chemical components were related to animal activity because higher concentrations of most components were observed when lights were on than during overnight periods. Volatile fatty acids, a component of odor, were only detected in the particulates not the gas phase, indicating that particulates may be the carrier of odor for some compounds rather than gases. This baseline study gives information for stakeholders that the most important tactic for reducing particulate generation and odor is to reduce animal activity.

Review Publications
Agga, G.E., Galloway, H.O., Netthisinghe, A.M. 2022. Effects of age and pasture type on the concentration and prevalence of tetracycline and macrolide resistant Enterococcus species in beef cow-calf production system. Frontiers in Antibiotics. 1:1052316. https://doi.org/10.3389/frabi.2022.1052316.
Correndo, A.A., Pearce, A., Bolster, C.H., Spargo, J., Osmond, D., Ciampitti, I.A. 2023. The soiltestcorr R-package: a reproducible approach for correlation analysis of crop yield and soil test data. SoftwareX. volume 21. https://doi.org/10.1016/j.softx.2022.101275.
Bolster, C.H., Correndo, A.A., Pearce, A., Spargo, J., Slaton, N., Osmond, D. 2022. A spreadsheet for determining critical soil test values using the modified arcsine-log calibration curve. Soil Science Society of America Journal. 87(1):182-189. https://doi.org/10.1002/saj2.20498.
Kaiser, R.A., Polk, J., Datta, T., Keely, S., Brinkman, N., Parekh, R.R., Agga, G.E. 2023. Occurrence and prevalence of antimicrobial resistance in urban karst groundwater systems based on targeted resistome analysis. Science of the Total Environment. 874. Article 162571. https://doi.org/10.1016/j.scitotenv.2023.162571.
Ruiz-Aguilar, G.M., Nunez-Palenius, H.G., Lovanh, N.C., Camarena-Martinez, S. 2022. Comparative study of methane production in a one-stage vs. two-stage anaerobic digestion process from raw tomato plant waste. Energies. 15(23). Article 9137. https://doi.org/10.3390/en15239137.
Agga, G.E., Kaiser, R., Polk, J., Allard, M. 2023. Prevalence and whole-genome sequencing characterization of Salmonella in urban karst groundwater and predominantly groundwater-fed surface waters for serotypes and antimicrobial resistance. Journal of Environmental Quality. 52(3):691-705. https://doi.org/10.1002/jeq2.20470.
Gutema, F.D., De Zutter, L., Piérard, D., Hinckel, B., Imamura, H., Rasschaert, G., Abdi, R.D., Agga, G.E., Crombé, F. 2023. Core genome sequencing analysis of E. coli O157:H7 unravelling genetic relatedness among strains from cattle, beef and humans in Bishoftu, Ethiopia. Microbiology Research. 14(1):148-160. https://doi.org/10.3390/microbiolres14010013.
Agga, G.E., Galloway, H., Appala, K., Mahmoudi, F., Kasumba, J., Loughrin, J.H., Conte, E. 2023. Effect of continuous in-feed administration of tylosin to feedlot cattle on macrolide and tetracycline resistant enterococci in a randomized field trial. Preventive Veterinary Medicine. 215. Article 105930. https://doi.org/10.1016/j.prevetmed.2023.105930.
Agga, G.E., Galloway, H.O., Netthisinghe, A.M., Schmidt, J.W., Arthur, T.M. 2022. Tetracycline-resistant, third-generation cephalosporin–resistant, and extended-spectrum b-lactamase–producing Escherichia coli in a beef cow-calf production system. Journal of Food Protection. 85(11):1522-1530. https://doi.org/10.4315/JFP-22-178.
Bedane, T., Agga, G.E., Gutema, F.D. 2022. Hygienic assessment of fish handling practices along production and supply chain and its public health implications in Central Oromia, Ethiopia. Scientific Reports. 12:13910. https://doi.org/10.1038/s41598-022-17671-5.
Netthisinghe, A., Galloway, H., Agga, G.E., Gunter, P., Sistani, K.R. 2023. Effects of cropping systems on soil physicochemical properties and abundances and spatial distributions of nitrogen-cycling bacteria. Agronomy. 13(6). Article 1461. https://doi.org/10.3390/agronomy13061461.
Gelalcha, B.D., Brown, S., Crocker, H., Agga, G.E., Kerro Dego, O. 2022. Regulation mechanisms of virulence genes in enterohemorrhagic Escherichia coli. Foodborne Pathogens and Disease. 19(9). https://doi.org/10.1089/fpd.2021.0103.
Marami, L.M., Berhanu, G., Tekle, M., Agga, G.E., Beyene, T.J., Tufa, T.B., Beyi, A.F., Edao, B.M. 2022. Antimicrobial resistance of staphylococci at animal human interface in smallholder dairy farms in Central Oromia, Ethiopia. Infection and Drug Resistance. 15:3767-3777. https://doi.org/10.2147/IDR.S370592.
Zeng, X., Vidlund, J., Gillespie, B., Cao, L., Agga, G.E., Lin, J., Kerro Dego, O. 2023. Evaluation of immunogenicity of enterobactin conjugate vaccine for the control of E. coli mastitis in dairy cows. Journal of Dairy Science. https://doi.org/10.3168/jds.2022-23219.
Loughrin, J.H., Silva, P.J., Lovanh, N.C., Sistani, K.R. 2022. Acoustic stimulation of anaerobic digestion: Effects on biogas production and wastewater malodors. Environments. 9(8). Article 102. https://doi.org/10.3390/environments9080102.
Silva, P.J., Cress, T., Drover, R., Michael, C., Docekal, G., Larkin, P., Godoy, A., Cavero, D., Sin, C., Waites, J., Mahmood, R., Cohron, M., Purvis-Roberts, K. 2023. Characterization of particle size distributions and water-soluble ions in particulate matter measured at a broiler farm. Agriculture. 13(7). Article 1284. https://doi.org/10.3390/agriculture13071284.
Gebremedhin, M., Coyne, M.S., Sistani, K.R. 2022. How much margin is left for degrading agricultural soils? The coming soil crises. Soil Systems. 6(1). Article 22. https://doi.org/10.3390/soilsystems6010022.