Location: Agricultural Water Efficiency and Salinity Research Unit
2023 Annual Report
Objectives
This project uses an integrated systems approach to identify dissemination of antibiotic resistant determinants (ARDs) in wastewater to soil, agricultural produce, and earthworms - linking production to consumption and environmental release, as well as conducting research on amendments and our novel mitigation technology that have the promise to reduce the environmental distribution of those determinants.
Objective 1: Identify the potential transmission routes of antibiotics and ARGs from manure and wastewater to soil-plant-earthworm systems for elucidation of key components for developing mitigation strategies.
Sub-objective 1A: Identify the role of agricultural produce, earthworms, and endophytic microbes in the dissemination of AMR in the agricultural environment/food chains.
Sub-objective 1B: Transfer of antibiotics and ARDs through food chains using a whole-system approach under outdoor conditions.
Sub-objective 1C: Determine concentrations of antibiotics and ARDs in the above and below ground edible portions of the plants being grown under nearly natural conditions.
Objective 2: Evaluate the risk associated with the potential dissemination of antibiotics, pathogens, and antimicrobial resistance through the natural ecological food chain-soil-plant-earthworm continuum and in association with relevant food production systems.
Objective 3: Assess the use of biochar application to soil as a mitigation strategy to limit the dissemination of antimicrobial resistance from soil to plants.
Sub-objective 3A: Explore the effects of biochar amendment on ARDs availability in soil and uptake in plants and earthworms.
Sub-objective 3B: Measure the effects of biochar on mitigation of ARDs under field conditions.
Objective 4: Develop a system for removal of antibiotics and other chemicals of emerging concern (CECs) from wastewater by passage through various layers of environmental media. Mitigation of the dissemination of antibiotic resistance through agricultural systems is best served by preventing the release of antibiotics and ARDs into such systems. Therefore, simple, cost-effective treatment systems to remove these CECs from treated wastewater are required, prior to the use of such wastewater for agricultural irrigation. We have recently developed a layered system of environmental media (Figure 3), which has shown the potential for removing antibiotic compounds from wastewater (Ashworth and Ibekwe, 2020). This system would be further developed under the current proposal.
Sub-objective 4A: Assess various environmental media in terms of their removal of CECs from wastewater.
Sub-objective 4B: Quantify the potential for the materials identified under Sub-objective 4A to remove CECs from a layered system based on modeling studies.
Sub-objective 4C: Assess layered systems (at various experimental scales) comprised of these environmental media to determine their effectiveness in CEC removal.
Approach
The research will be conducted to:
Objective 1: Identify the potential transmission routes of antibiotics and ARGs from manure and wastewater to soil-plant-earthworm systems for elucidation of key components for developing mitigation strategies. The work will be conducted using greenhouse (objective1a), outdoor (large- scale) pot (objective1b), and lysimeter experiments (objective1c), which are of a sufficiently large scale to allow for natural biological processes to take place, while still being highly controllable. In the pot studies, we will assess changes in soil microbial composition as well as concentrations of antibiotic compounds, and identify ARDs in the soil, soil solution, rhizosphere, phyllosphere, and earthworm gut in response to wastewater irrigation. The experiment will inform more realistic and integrated studies conducted using intermediate (40-liter pots) and large scale (lysimeter) experiments to assess time-course trends in the transfer of antibiotics and ARDs through food chains using a whole-system approach. The results of these studies will be used to assess the potential risk of antibiotic resistance dissemination by evaluating bioaccumulation/biomagnification factors of AT/ARGs dissemination in the food chain (Objective 2).
Objective 3: Assess the use of biochar application to soil as a mitigation strategy to limit the dissemination of antimicrobial resistance from soil to plants. Since biochar has been shown to effectively mitigate CEC transport, it will be assessed as a mitigation strategy to reduce the dissemination of antibiotic resistance. A greenhouse pot and field experiments will be conducted using the most promising of the biochar materials to quantify any potential mitigation effect in terms of antibiotic and ARD dissemination in the environment. This pot experiment will use soils applied with agriculturally relevant rates of biochar (e.g., 0.1, 0.5 and 1% by mass; equivalent to 2.6, 13, and 26 t/ha, respectively), while the field experiment will measure the impact of biochar application on the dissemination of antibiotics and ARDs from wastewater and manure to soil-plant-earthworm continuum. One limitation in the field work may be low gene targets for qPCR for monitoring of ARGs. Here, we will adopt droplet digital PCR (dd- PCR) if we identify low concentrations using qPCR that reduces reproducibility and efficiencies of qPCR.
Objective 4: Develop a system for removal of antibiotics and other chemicals of emerging concern (CECs) from wastewater by passage through various layers of environmental media. The final phase of this work will focus on developing a system for the removal of CECs, ARB, and ARGs from wastewater using bioreactors that enhance different bioprocesses to reduce the different classes of CEC and biological determinants. Layered system of environmental media consisting of gravel, sand, soil, and soil+biochar will be used to remove antibiotic compounds from wastewater. This system will be developed, tested, scale-up, and modeled (Hydrus 1-D) for removal of antibiotics and other chemicals of emerging concern from wastewater by passing through the various media.
Progress Report
This report documents fiscal year (FY) 2023 progress for project 2036-12320-011-000D, titled, “Protection of Food and Water Supplies from Pathogens and Human Induced Chemicals of Emerging Concern”.
In support of Objective 1, ARS researchers form Riverside, California, carried out manure and treated wastewater analyses have been carried out to identify constituent antibiotic compounds and their concentrations. In addition, an outdoor pot experiment was conducted to examine the relationships between (i) antibiotic inputs from treated municipal wastewater and manure and (ii) the development and spread of antibiotic resistance determinants (ARDs), such as antibiotic resistant bacteria and antibiotic resistance genes, in the soil-plant-earthworm continuum. In these experiments, spinach and radish plants were grown in soils treated with antibiotic-laden manure and/or antibiotic-laden treated municipal wastewater. Earthworms (Eisenia fetida) were also introduced into the soil. The fate and transport (plant uptake, leaching, soil adsorption, degradation, earthworm uptake) of the antibiotics are being determined and will be considered in relation to the development and dissemination of ARDs. The analyses of samples and data from these experiments are ongoing.
ARS researchers also made progress in support of Objectives 3 and 4 through the characterization and application of biochar materials within the larger framework of the project plan. Biochar materials for Objective 3 were acquired and characterized on the basis of several functionally-relevant parameters, including Cation Exchange Capacity (CEC), pH, elemental composition and ash/volatile/moisture content. These parameters are key to understanding the role of biochar in mitigating the uptake and transport of antibiotics in Objective 3 experiments. Objective 4 was further addressed through probing the influence of a key wastewater constituent (dissolved organic matter (DOM)) on antibiotic adsorption from solution by biochar and clay. It was shown that DOM binds the antibiotic cephalexin in solution and reduces cephalexin adsorption by clay and biochar. Reduction of adsorption by DOM was lower on clay than on biochar. These results inform the behavior of antibiotics in DOM-rich wastewater streams and how they may impact antibiotic adsorption by biochar materials implemented in this project. Additional preliminary studies have been conducted probing the influence of biochar oxidation on antibiotic adsorption. As biochar materials are applied in soils or column type adsorption systems, they may become increasingly oxidized. To determine the impact this may have on long-term antibiotic immobilization, adsorption studies with biochars and oxidatively aged biochars are underway. Preliminary results show oxidative aging influences antibiotic adsorption dynamics and may lead to decreased antibiotic adsorption affinity and capacity.
In further support of Objective 4, the efficacies of various engineered biochars (identified under Objective 3) at removing antibiotics from water under dynamic conditions were quantified. This work has highlighted correlations between biochar characteristics (determined under Objective 3) and antibiotic retention, which has important implications for the selection of appropriate biochar materials for such treatment systems. This work has also indicated the potential importance of using dynamic (flow-through) approaches, rather than batch equilibrium approaches, to quantify antibiotic retention. In supplementary ARS-funded work (Antimicrobial Resistance/Alternatives to Antibiotics; AMR/ATA funding) related to Objectives 3 and 4, effluents from biochar-based treatment systems are being used to irrigate soils containing common earthworms to assess the efficacy of the systems at preventing the development of antibiotic resistance in soils and the earthworm gut.
Accomplishments
1. Dissemination of Antimicrobial Resistance in the Food Production Environment. The increasing demand for food production and the expansion of irrigated agriculture have put significant pressure on water resources, particularly in arid and semi-arid regions. Identifying and developing effective methods for tracking the dissemination of antimicrobial resistance in the food production environment is a recent goal of many International and National Organizations. ARS researchers in Riverside, California, working in collaboration with University of California at Riverside researchers, used metagenomics to identify the number of genes and antibiotic-resistant bacteria enriched in spinach and radish. Researchers found that the number of antibiotic-resistant bacteria was significantly higher in spinach compared to radish, and there was a strong correlation between genes and microbial diversities in the spinach and radish production environment. Thus, changes in the abundance of antibiotic resistance genes may exert selective pressure on the microbial community, influencing its composition and diversity. This study provides new knowledge to assist farmers, researchers, and wastewater management professionals in reducing the dissemination of antimicrobial resistance in agricultural systems, ultimately protecting the public from the threat of antimicrobials.
Review Publications
Ashworth, D.J., Ibekwe, A.M., Men, Y., Ferreira, J.F. 2022. Dissemination of antibiotics through the wastewater–soil–plant–earthworm continuum. Science of the Total Environment. 858. Article 159841. https://doi.org/10.1016/j.scitotenv.2022.159841.
Schmidt, M.P., Ashworth, D.J., Marquez Celis, N., Ibekwe, A.M. 2023. Optimizing date palm leaf and pistachio shell biochar properties for antibiotic adsorption by varying pyrolysis temperature. Bioresource Technology Reports. 21. Article 101325. https://doi.org/10.1016/j.biteb.2022.101325.
Ibekwe, A.M., Bhattacharjee, A.S., Phan, D., Ashworth, D.J., Schmidt, M.P., Murinda, S.E., Obayiuwana, A., Murry, M.A., Schwartz, G., Lundquist, T., Ma, J., Karathia, H., Fanelli, B., Hasan, N.A., Yang, C. 2023. Potential reservoirs of antimicrobial resistance in livestock waste and treated wastewater that can be disseminated to agricultural land. Science of the Total Environment. 872. Article 162194. https://doi.org/10.1016/j.scitotenv.2023.162194.
