Research Project: Developing Safe, Efficient and Environmentally Sound Management Practices for the Use of Animal Manure

Location: Food Animal Environmental Systems Research

2021 Annual Report

Objectives
Obj 1: Enhance the benefits of using animal manure as means of improving soil productivity for longterm production systems. 1.1 Improve soil health/quality through a longterm mngmnt practice involving animal manure, cover crop, & crop rotation. 1.2 Determine if poultry litter application timing (fall/spring) & cover crop (cover crop/no cover crop) affects on corn N use efficiency & yield. 1.3 Develop improved models for describing phosphorus cycling in soils amended with animal manure. 1.4 Develop an energy-efficient & cost-effective method of capturing ammonia from animal facilities & subsequent usage as fertilizer. 1.5 Evaluate poultry litter application on the incidence of Clostridium botulinum in haylage forage harvest systems & stand persistence. 1.6 Improve sustainability through a management strategy using cover crop grazing & animal manure to improve soil quality & fertility while increasing grain & livestock production. 1.7 Combining a multi-proxy investigation of agricultural contaminant transport in Karst groundwater systems & informal education to improve stewardship & best management practices. Obj 2: Develop strategies for managing the survival & transport of antibiotics, pathogens & indicator species through agroecosystems. 2.1 Determine whether biochar-amended sand filters are an effective management practice for removing pathogenic & indicator microorganisms from tile drainage waters. 2.2 Characterize persistence and transmission of antibiotics, antibiotic resistance (AR) genes, AR mobile genetic elements and AR bacteria through agro-ecosystems. 2.3 Determine factors controlling adhesion & survival of manure-borne pathogens in manured soils & on crops & identify mitigation strategies. 2.4 Develop of methods for measuring antibiotics in agricultural samples & the correlation of the presence of antibiotic resistant bacteria. 2.5 Develop a strategy for rapid detection & deletion of antibiotic resistance genes using DNA-binding domain based nucleases. Obj 3: Assess gaseous & particulate matter emissions resulting from animal prod. facil. & manure application sites. 3.1 Develop methods for the determination of VOC that contribute to malodorous emissions from field applied manures & use these methods to compare emissions from different manure application methods to field crops. 3.2 Obtain field measurements of the temporal variability of gas & particulate matter emissions. 3.3 Determine the fate of atmospheric N and sulfur emissions from agricultural systems. 3.4 Measure & model the fates of VOCs, PM, & GHGs from agricultural systems. 3.5 Model the chemical partitioning of N compounds between gas & particulate phase. Obj 4: Improve production efficiency & emissions management of renewable bioenergy production systems using technologies such as anaerobic digesters. 4.1 Investigate biogas production and substrate utilization of poultry litter & use microaeration to improve biogas production. 4.2 Optimize anaerobic digestion of poultry wastes to reduce emissions & obtain bioenergy for sustainability. 4.3 Develop novel & cost-effective composite alginate filter materials to capture ammonia, GHG, odors from livestock prod. facil.

Approach
This project was conceived as a cooperative/partnership and comprehensive research program between ARS and Cooperator. The project is designed to utilize the scientific expertise and facilities of both institutions to conduct problem-solving research related to animal waste management in Kentucky and the southeastern U.S. Through a series of innovative, cooperative research experiments at the animal production facilities and manure application sites, we will investigate manure microbial and nutrient losses as gas and through runoff water and odorous compounds from land and animal production sites. We will also determine levels of these compounds/contaminants in manure, application sites, and loading rates in agroecosystems. We will also investigate and find solutions to the environmental and agronomic problems such as nutrients, pathogens, greenhouse gases (GHGs), ammonia, odor-causing VOCs, particulates, dust, and sediment associated with animal production facilities and manure application lands. This will be accomplished based on new knowledge gained through the multidisciplinary approaches and the expertise of the scientists at this location through basic and applied research in the laboratory and in real farming sites and animal production environments. We will use the research results and develop best management practices (BMPs) for crop production on land receiving animal manure with regard to manure application timing, rate, nutrient, and loss of hazardous compounds within the agroecosystem of the unique “karst topography” common to this region. The research effort will be multi-disciplinary and multifaceted in support of decision making and systems development. Information generated from this research will be integrated into regional and national databases and statistical models to contribute towards improved environmental quality, sustainability, and economic viability of farms.

Progress Report
Laboratory analysis is ongoing to evaluate the impact of organic fertilizer as soil amendment on the antimicrobial resistance of bacteria cultured from soil samples. Longitudinal experiment consisting of poultry litter, commercial fertilizer and untreated control plots was conducted on cornfield for 172 days post treatment applications spanning the entire corn cropping and growing season. A total of 576 soil samples were collected on six occasions. Samples were cultured for quantification and detection of generic-, tetracycline resistant- and third generation cephalosporin resistant- Escherichia coli, generic-, tetracycline resistant- and macrolide resistant- Enterococcus species, extended spectrum beta-lactamase producing bacteria, carbapenem resistant enterobacteria. These bacteria are being confirmed by polymerase chain reaction (PCR) and targeting specific DNA sequences and characterized for their antimicrobial resistance using susceptibility testing and by PCR to detect genes responsible for resistance. Two manuscripts are planned from this sub-objective. Laboratory analysis is underway for Subobjective 2.3 to evaluate the impact of continuous in-feed use of tylosin, an antimicrobial compound, in feedlot cattle on the antibiotic resistance of bacteria isolated from the fecal samples of cattle. A cohort of weaned calves was randomized at arrival at feedlot to receive tylosin medicated feed (10 calves) or non-medicated feed (10 calves). Animals were followed for about a year during which feces from the rectum, manure from pen-surface and animal feed were collected and cultured for antibiotic resistant Enterococcus species and E. coli. Bacterial isolates are being confirmed by PCR and further characterized for antibiotic resistance using susceptibility testing and PCR to detect genes responsible for resistance. Two manuscripts are planned from this subobjective. The Western Lake Erie Basin (WLEB) has experienced significant adverse effects from eutrophication that have negatively affected the commercial fishing industry and forced the City of Toledo to shut down its water treatment plant for three days in 2014. Phosphorus loss from tile-drained agricultural fields has been identified as a significant contributor to WLEB eutrophication. Working with ARS researchers in Columbus, Ohio, we are evaluating the use of simple mathematical models for describing the loss of phosphorus from tile-drained fields within the WLEB. Models are an important tool to help landowners, nutrient management specialists, and regulatory agencies better direct limited resources towards best management practices that are most appropriate for the dominant sources and pathways of P loss. Preliminary results indicate that using published values of runoff parameters for a commonly used surface runoff model are not applicable to the tile-drained fields within the WLEB. Research is ongoing to test alternative methods for estimating model parameters for describing surface and tile runoff in these fields. An experiment designed to test the adaptation of methanogenic consortia to alkaline conditions were started. In these experiments, anaerobic digesters were seeded with dairy manure and then fed dairy manure with increasing concentrations of inorganic bases to test how alkaline conditions affect biogas quality and production as well as microbial community structure. It is hypothesized that gradually increasing the alkalinity of anaerobic digestate will facilitate microbial adaptation and cause shifts in microbial community structure, lower biogas carbon dioxide concentration through bicarbonate sequestration, and shift methane production away from acetoclastic methanogenesis towards syntrophic acetate oxidation and hydrogenotrophic methanogenesis using bicarbonate as an electron acceptor for methane production. If positive results are obtained, this could indicate a means of operating highly stable anaerobic digesters with greatly increased biogas quality and reduced sulfur emissions. A building to house two pilot scale anaerobic digesters was constructed and rebuilding of the anaerobic digesters and wastewater treatment systems has begun. The wastewater treatment system will be used to produce biogas through sound-assisted breakdown of manure sludge. Effluent from the treatment system will be treated to remove phosphorus and nitrogen and discharged into an aerobic lagoon. Water from the lagoon will be mixed with manure and fed back to the treatment system. Except for manure inputs, the systems will operate largely as a closed loop treatment system with little or no discharge to the environment. Continuing field study in monitoring and optimization of methane production from an anaerobic digestion system (15 million gallons) of poultry litter mixed with food wastes and evaluation of different lignocellulosic enzymes on biogas production from recalcitrant livestock waste feedstocks. The optimization of nanoparticles formation from plant extracts was also studied to examine its 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. Under Sub-objective 3.2, data analysis on gas and particulate data from all field sites are completed. One modeling paper of poultry houses is published. Two papers are submitted on sampling. Three other papers being prepared on gas and particulate data from a poultry house, a mink house, and comparing several different animal operations. Under Sub-objective 3.3, Analysis of the data from all experiments at the University of California, Riverside is complete. One publication is accepted and published. Another is submitted. Two more are under preparation. Research activities continued on two field experiments under Sub-objective 1, the crop rotation, cover crops, and timing of manure subsurface application are being studied to investigate how maximizing the soil health management practices can improve soil’s physical, biological, and chemical properties. Cover crops were rolled down prior to the planting of the next rotation crop. Data are being analyzed for presentations and manuscript preparation for publication in the scientific journals.

Accomplishments
1. In situ sonification of anaerobic digestion: Extended evaluation of performance in a temperate climate. Increasing the efficiency of wastewater treatment and improving sludge breakdown is vital to reducing the cost of biogas production and reducing environmental costs of sludge disposal. ARS researchers at Bowling Green, Kentucky, compared two unheated wastewater treatment systems, one using sound supplied by waterproofed speakers and one with no sound, for over a year. The wastewater was treated in an air free environment and discharged wastewater was mixed with feed and fed back to the system. Wastewater is typically saturated with gases due to the digestion and these gases form bubbles that vibrate in response to sound. These vibrations can be used to speed up the breakdown of wastewater. Exposure of the gas saturated wastewater to a low frequency sound induced bubbles to produce harmonics up to, and beyond, the range of human hearing. This result shows that bubbles can be used to treat the wastewater more effectively to sound than is the case with ultrasonic treatment which is absorbed at very short distances. Through the summer, the sound-treated system produced 27% more gas than the control system, and 74 times more during the winter when gas production by the control system stopped. Afterwards, the control system produced more gas due to depletion of feed in the sound-treated digester. Results show that sound can be used for faster digester startup and substitute for a share of heating requirements during cool months. This will impact and benefit livestock producers and farmers.

2. Evaluation of microaeration and sound to increase biogas production from poultry litter. Small amounts of air introduced into otherwise airless wastewater, has been shown to enhance methane production. This occurs by promoting the growth of bacteria that tolerate small amounts of oxygen and that can digest substances that oxygen sensitive bacteria cannot. The treatment of wastewater with sound has also been shown to increase methane gas production. Air treatment and treatment with sound as well as combined air and sound were compared by ARS researchers at Bowling Green, Kentucky, to an untreated digester for the production of gas and if they could improve wastewater quality. Chicken waste from a farm using wood chips as floor covering was used. At first, the digesters were fed 400 grams per week, and this was slowly increased to 2400 grams per week. Compared to untreated wastewater, sound treatment, air, and sound/air together produced more gas. The air only treatment may have worked better than combined air and sound due to the sound causing escape of air from the digesters. Digesters treated with sound had the highest amounts of particles, likely due to air bubbles attaching to the particles and lifting them. This will impact and benefit livestock producers and farmers.

3. Optimization of nanoparticles formation from plant extracts and their effectiveness on agricultural pathogens. Antibiotic resistant microorganisms are on the rise in the environment due to the increasing use and/or inappropriate use of antibiotics in human medicine and livestock production. It is necessary to find a simple and economical way to reduce the proliferation of these antibiotic resistant microorganisms. Nanoparticles with antimicrobial properties along with medicinal plants hold a great promise in this arena, especially nanoparticles that can be synthesized from eco-friendly methods. ARS scientists from Bowling Green, Kentucky, along with researchers from universities in South Korea and Mexico carried out the optimization of nanoparticle synthesis mediated from plant extracts as a model to utilize. These nanoparticle formations were optimized based on the right ratios of extracts and metallic concentrations. The results show that the maximum formation concentrations were obtained at almost equal ratios of extract and metallic concentrations. These nanoparticles are also effective against agricultural pathogens such as Escherichia coli. Synthesized nanoparticles have increased potency against E. coli in optimum culture media in comparison to metallic ions alone. For example, silver nanoparticles treated microcosms achieved 100% killed in less incubation time and lower dosage than bulk silver ion. Based on these findings, the current method can be suitable for the scale-up production of nanoparticles from a commonly available plants in a fight against agricultural pathogens that could harbor antibiotic resistance genes.

4. Governing microbial transport through biochar-amended sand filters. Animal manure is often applied to agricultural fields to add nutrients and organic matter to the soil to enhance soil quality and fertility. Manure, however, can also be a source of pathogenic microorganisms that pose a threat to humans. Because tile drainage significantly alters field hydrology, when manure is applied to tile-drained fields the movement of pathogens from agricultural fields to drinking water can be greatly enhanced. An ARS research at Bowling Green, Kentucky, investigated the potential of using biochar – a charcoal-like material generated during the pyrolysis of biomass – as an amendment to sand filters as a management strategy for removing pathogenic and indicator microorganisms from tile-drainage waters. Laboratory experiments were conducted to determine the best combination of biochar concentration and sand grain size for maximizing both retention capacity of bacteria and water flow. Results indicate that the addition of the biochar reduces the transport of E. coli and Salmonella through laboratory-scale biofilters and that the amount of retention is strongly dependent on biochar application rate. Results also show that sand size is a strong determinant on the ability of the biochar to retain bacteria, with retention decreasing with increasing grain size. Research also showed that treating biochar with Fe-hydroxides can modestly increase retention of bacteria while also drastically increasing retention of phosphorus, a nutrient that can lead to water quality problems and is often found to be elevated in tile-drainage waters. Results from this research can help guide the development and application of using biochar-amended sand filters for treating tile-drainage waters prior to release into the environment.

5. Modeling of sulfur emissions from animal operations. Sulfur is emitted from animal operations as reduced sulfur compounds and can react in the atmosphere to produce sulfur dioxide and particulate matter. As sulfur emissions from coal power plants continues to decline, agriculture-related sulfur is expected to elicit more scrutiny, but modeling of agriculture-emitted sulfur compounds has not typically been performed to understand the impact. Sulfur emissions and its dispersion over the southeastern U.S. were simulated by ARS researchers at Bowling Green, Kentucky, using the Weather Research and Forecasting-Chemistry model. Simulations were performed for dry periods and precipitation events. Spatial coverage of sulfur dispersion was reduced during precipitation events and expanded during dry periods. The highest concentrations of sulfur remained within close range of the emission locations for the majority of the simulations, except for when local surface wind speeds were high. Most emissions from the locations remained limited to the surface and lower levels of the atmosphere. This has impact on the scientific community and regulatory bodies to correctly model and assess the effects of agricultural emissions to air quality.

6. Antibiotic and metal resistance genes increased following anaerobic digestion of tetracycline containing animal manure. Antibiotics are used for the treatment of diseases in food producing animals. Metals such as copper and zinc are essential in animal nutrition. Antibiotics, metals, and bacteria including resistant ones are excreted through feces and urine resulting in environmental pollution. Once excreted, antibiotics exert selection pressure on native soil bacteria leading to propagation and spread of antibiotic resistant bacteria contaminating surface waters through run-offs from animal manure amended soils. Microbial digestion of animal manure in the absence of oxygen, while also producing renewable energy, is a promising technology for the management of animal manure. ARS scientists in Bowling Green, Kentucky, in collaboration with researchers from Western Kentucky University, investigated the effect of anaerobic digestion on the removal of antibiotic and metal resistance genes, and bacteria in cattle, poultry and swine manure. Manure samples were spiked with tetracycline antibiotics and digested for 64 days. While anaerobic digestion decreased the concentration of tetracyclines but not completely removed, it increased the concentrations of tetracycline and methicillin resistance genes, total bacterial population including Escherichia coli, enterococci, and Staphylococcus aureus species. Anaerobic digestion did not have any impact on the concentrations of metals themselves, and concentrations of metal resistance genes either increased or remained unaffected depending on the animal species. This study showed the need for post-digestion treatments of animal manure to remove bacteria, antibiotic resistance genes, heavy metals, and their resistance genes to reduce environmental and public health impacts. This has impact on the scientific community, farmers, and local and federal government regulators.

7. Atmospheric chemistry and secondary product analysis of reduced sulfur emissions. Several sulfur compounds emitted from animal operations are known precursors to particulate matter formation in the atmosphere from previous studies of the same compounds in the marine atmosphere due to ocean emissions. Laboratory experiments in a Teflon environmental chamber were conducted by ARS researchers at Bowling Green, Kentucky, to study oxidation and particulate forming potential of several sulfur compounds. Experiments were conducted with both the presence and absence of other urban pollutants to observe interactions. One expected product, methanesulfonic acid, was never observed during the study in the presence of other emissions. The absence of methanesulfonic acid when in the presence of other emissions has not been reported in previous studies and indicates a lack of understanding of the atmospheric chemistry. It implies that chemistry observed over the continent in agriculture regions may diverge from what has been observed in ocean regions. This information is important for the scientific and public health communities to help them correctly estimate impacts of agricultural activities to the atmosphere.

Review Publications
Winchester, J., Mahmood, R., Rodgers, W., Silva, P.J., Lovanh, N.C., Durkee, J., Loughrin, J.H. 2021. A model-based exploratory study of sulfur dioxide dispersions from concentrated animal feeding operations in the Southeastern United States. Physical Geography. https://doi.org/10.1080/02723646.2021.1875583.
Netthisinghe, A., Galloway, H., DeGraves, F., Agga, G.E., Sistani, K.R. 2020. Grain yield and beef cow–calf growth performance in dual-Purpose and conventional grain wheat production systems and stockpiled tall fescue pasturing. Agronomy Journal. 10(10). Article 1543. https://doi.org/10.3390/agronomy10101543.
Amenu, K., Agga, G.E., Kumbe, A., Shibiru, A., Desta, H., Tiki, W., Kerro Dego, O., Wieland, B., Grace, D., Alonso, S. 2020. Milk symposium review: Community-tailored training to improve the knowledge, attitudes, and practices of women regarding hygienic milk production and handling in Borana pastoral area of southern Ethiopia. Journal of Dairy Science. 103(11):9748-9757. https://doi.org/10.3168/jds.2020-18292.
Aregawi, W.G., Gutema, F., Tesfaye, J., Sorsa, A., Megersa, B., Teshome, P., Agga, G.E., Ashenafi, H. 2020. Efficacy of diminazene diaceturate and isometamidium chloride hydrochloride for the treatment of Trypanosoma evansi in mice model. Journal of Parasitic Diseases. 45:131-136. https://doi.org/10.1007/s12639-020-01289-3.
Agga, G.E., Silva, P.J., Martin, R.S. 2020. Third-generation cephalosporin- and tetracycline-resistant escherichia coli and antimicrobial resistance genes from metagenomes of mink feces and feed. Foodborne Pathogens and Disease. 18(3):169-178. https://doi.org/10.1089/fpd.2020.2851.
Gutema, F.D., Agga, G.E., Makita, K., Smith, R.L., Mourits, M., Tufa, T.B., Leta, S., Beyene, T.J., Asseffa, Z., Urge, B., Ameni, G. 2020. Evaluation of options to control bovine tuberculosis in Ethiopia using multi-criteria decision analysis. Frontiers in Veterinary Science. 7. Article 586056. https://doi.org/10.3389/fvets.2020.586056.
Gutema, F.D., Rasschaert, G., Agga, G.E., Merera, O., Duguma, A.B., Abdi, R.D., Duchateau, L., Mattheus, W., Gabriel, S., De Zeutter, L. 2021. Prevalence, antimicrobial resistance, and molecular characterization of salmonella in cattle, beef, and diarrheic patients in Bishoftu, Ethiopia. Foodborne Pathogens and Disease. 18(4):283-289. https://doi.org/10.1089/fpd.2020.2869.
Woyessa, M., Agga, G.E., Gumi, B., Ayana, D., Mamo, G. 2020. Antibiotic use in poultry production in selected districts of east Showa zone, central Ethiopia: from antibiotic stewardship perspective. American-Eurasian Journal of Scientific Research. 15(3):101-111.
Agga, G.E., Kasumba, J., Loughrin, J.H., Conte, E.D. 2020. Anaerobic digestion of tetracycline spiked livestock manure and poultry litter increased the abundances of antibiotic and heavy metal resistance genes. Frontiers in Microbiology. 11. Article 614424. https://doi.org/10.3389/fmicb.2020.614424.
Gutema, F.D., Yohannes, G.W., Abdi, R.D., Abuna, F., Ayana, D., Waktole, H., Amenu, K., Hiko, A., Agga, G.E. 2020. Dipylidium caninum infection in dogs and humans in Bishoftu town, Ethiopia. Diseases. 9(1). https://doi.org/10.3390/diseases9010001.
Agga, G.E., Silva, P.J., Martin, R.S. 2021. Detection of extended-spectrum beta-lactamase-producing and carbapenem-resistant bacteria from mink feces and feed in the United States. Foodborne Pathogens and Disease. 18(7): 497-505. https://doi.org/10.1089/fpd.2020.2898.
Balemi, A., Gumi, B., Amenu, K., Girma, S., Gebru, M., Tekle, M., Agga, G.E., Rius, A., D'Souza, D., Kerro Dego, O. 2021. Prevalence of mastitis and antibiotic resistance of bacterial isolates from cmt positive milk samples obtained from dairy cows, camels, and goats in two pastoral districts in southern Ethiopia. Animals. 11(6). Article 1530. https://doi.org/10.3390/ani11061530.
Gutema, F.D., Agga, G.E., Abdi, R.D., Alemnesh, J., Duchateau, L., De Zeutter, L., Gabriel, S. 2021. Assessment of hygienic practices in beef cattle slaughterhouses and retail shops in Bishoftu, Ethiopia: Implications for public health. International Journal of Environmental Research and Public Health. 18(5),2729. https://doi.org/10.3390/ijerph18052729.