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ARS Home Pacific West Area Riverside, California Agricultural Water Efficiency and Salinity Research Unit Research





- Ibekwe, Abasiofiok - Mark
- Ashworth, Daniel
- Schmidt, Michael - Mike
- 2023
- 2022
- Publications
- Soil and Air (212)

Research Project: Protection of Food and Water Supplies from Pathogens and Human Induced Chemicals of Emerging Concern **Location:
                            Agricultural Water Efficiency and Salinity Research Unit** 2022 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 progress for project 2036-12320-011-000D, Protection of Food and Water Supplies from Pathogens and Human Induced Chemicals of Emerging Concern, which started October 2021 and continues research from projects 2036-32000-005-000D, Identifying, Quantifying and Tracking Microbial Contaminants, Antibiotics and Antibiotic Resistance Genes in Order to Protect Food and Water, and 2036-12130-011-000D, Predicting and Reducing Agricultural Contaminants in Soil, Water, and Air.

Under Objective 1, a greenhouse study was conducted to track the dissemination of antibiotic resistance genes and bacteria from treated wastewater used for irrigation through soil-plants-earthworm continuum. Two vegetable crops, spinach, and radish were grown and irrigated with treated wastewater, and earthworms were fed harvested plant materials. Genomic DNA was extracted from these samples to determine total bacterial composition, their resistance genes, and mobile genetic elements. Quantification of antibiotic resistance genes in bacterial communities and in bacteria is in progress. The transfer of antibiotic resistance determinants will be considered through the same continuum and to use more heavily contaminated wastewaters in which contaminants are already present in the water.

In support of Objectives 3 and 4, progress has been made in the areas of biochar production, modification, characterization, and application for adsorbing antibiotics. These processes are critical for assessing the role of biochar amendments to mitigate the development and propagation of antibiotic resistant determinants in recycled wastewater irrigated soils. Different biochar materials were produced from four different agricultural by-product feedstocks (i.e., rice husks, dairy manure, pistachio shells and date palm leaves). Pistachio shell and date palm leaf feedstocks were modified during pyrolysis by heating to different temperatures. This resulted in an array of biochars with differing surface properties and affinities for a mixture of antibiotic compounds found in Southern California wastewater. Higher pyrolysis temperatures yielded biochars with most favorable adsorption properties for all antibiotics, with over 93% removal efficiency observed for all antibiotics at the highest temperature used. Dairy manure and rice husk biochars were modified by acid and base treatments after production. Acid and base treatment resulted in biochars with notably different pH values, which translated to tunable removal of antibiotics from water in both batch equilibrium and dynamic transport studies. Ongoing research is currently focused on a multilayered biochar-sand column filtration system for removing a mixture of six prevalent antibiotics from water. The system consists of three layers of biochar with distinct antibiotic removal behavior combined with sand to enhance hydraulic properties. Preliminary results are showing excellent removal behavior, with column effluent showing non-detectable levels of antibiotics. In associated work, the application of these different biochars to soil has been found to mitigate the transfer of antibiotics from soil to earthworm tissues, i.e., reducing antibiotic bioavailabilty. The impact of this work is that it demonstrates the ability to transform common agricultural byproducts from California agroecosystems into a value-added product capable of mitigating potential antibiotic resistance risks associated with recycled wastewaters used for irrigation. This capability is critical for future efforts to maximize water use efficiency in water-stressed arid region agricultural systems across the western United States and worldwide. **Accomplishments**
                                

1. Culture methods for analyzing Salmonella with antimicrobial resistance in surface water. The presence of antimicrobial-resistant Salmonella enterica in water indicates environmental contamination and is an emergent food safety and public health issue. Identifying and developing effective and sensitive detection methods for Salmonella from surface water is a recent goal of the National Antimicrobial Resistance Monitoring System (NARMS), supported by Environmental Protection Agency (EPA), Food and Drug Administration (FDA), and USDA. ARS researchers at Riverside, California, Beltsville, Maryland, Athens, Georgia, Clay Center, Nebraska, and Lincoln, Nebraska, working in collaboration with NARMS partners FDA and EPA, have developed methods to compare and identify factors that affect recovery of antimicrobial resistant Salmonella from surface water. Each sample was subjected to one of three recovery and enrichment methods: Bulk Water enrichment (BW), vertical Modified Moore Swab (MM), and the modified Standard Method. Across all four locations, Standard Method was the method that most frequently recovered environmental and low levels of inoculated Salmonella from water, and all protocols have been uploaded to protocols.io and are accessible. The modified Standard Method more frequently recovered low levels of Salmonella from inoculated water samples and should be prioritized for Salmonella recovery from surface water in laboratory settings. 2. Agricultural byproducts converted to materials that remove antibiotics from water. As water resources globally become more stressed under a changing climate, recycled treated wastewater is receiving greater consideration as an irrigation water source. Recycled treated wastewater often contains trace levels of antibiotics, which, when applied to food crops, represents a potential risk for antibiotic resistance development in bacteria. This may subsequently threaten human and livestock health. Biochar, an environmentally friendly and cost-effective material produced from biomass, has shown promise for removing antibiotics from water, mitigating the introduction of antibiotics into agricultural systems. ARS researchers in Riverside, California, produced biochar materials from locally available date palm leaf and pistachio shell feedstocks under different conditions to study performance at removing antibiotics from water. They used high temperature pyrolysis (800 degrees C) to produce biochars capable of removing up to 99.5% of commonly occurring antibiotics from water. These materials will play an important role in the development of larger scale water treatment processes key to improving recycled water quality for irrigation use. 3. Agricultural byproducts converted to materials that limit the transfer of antibiotics from soil into earthworm tissues. As water resources globally become more stressed under a changing climate, recycled treated wastewater is receiving greater consideration as an irrigation water source. Recycled treated wastewater often contains trace levels of antibiotics, which, when applied to food crops, may lead to the transfer of antibiotics through food chains. This poses risks to human health as antibiotic resistance may then develop within plants and animals. Biochar, an environmentally friendly and cost-effective material produced by heating biomass, can potentially limit the biological availability of antibiotics in soil by strongly adsorbing the compounds. ARS researchers in Riverside, California, used a variety of biochars produced from different feedstocks under varying temperature conditions and post-production modifications to determine their effectiveness in limiting transfer of antibiotics from soil into earthworm tissues. Compared with no biochar addition to soil, the most effective biochars resulted in up to 90% reductions in antibiotic concentrations in earthworm tissues. Biochar materials are a potentially important resource for limiting the biological availability of antibiotics and the dissemination of antibiotic resistance in agricultural systems. **Review Publications**
                            *Obayiuwana, A., Ogunjobi, A., Ibekwe, A.M. 2021. Prevalence of antibiotic resistance genes in pharmaceutical wastewaters. Water. 13(13). Article 1731. https://doi.org/10.3390/w13131731.*
                                *Schwartz, G., Ibekwe, A.M., Lundquist, T., Murinda, S., Murry, M.A. 2021. Utilization of semi-continuous algae culture for the treatment of recycled dairy lagoon wash water. Current Biochemical Engineering. 7(1):72-82. https://doi.org/10.2174/2212711907666210622153521.*


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