Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521
Performing Department
(N/A)
Non Technical Summary
Agriculture accounts for more than 80% of the nation's water consumption. As global populations grow, more water will be required for agricultural production. Reclaimed water is highly treated wastewater that can be reused for various purposes, including agriculture and landscape irrigation. Using reclaimed water for agriculture will free up drinking water and increase our water supply reliability.Conventional municipal wastewater treatment plants produce reclaimed water by treating wastewater. These treatment plants receive wastewater containing various pollutants, including antibiotics and microorganisms. When these microorganisms are exposed to antibiotics, they can evolve antimicrobial resistance leading to an increase in antibiotic-resistance genes (ARGs), antibiotic-resistant bacteria (ARBs), and mobile genetic elements (MGEs). Antibiotics, ARGs, ARBs, and MGEs, are collectively known as antibiotic resistance determinants (ARDs). Wastewater treatment plants do not adequately remove ARDs; therefore, subsequent "polishing" to remove the ARDs from treated municipal wastewater is desirable to curtail antibiotic resistance in agricultural fields irrigated with reclaimed water and the environment.Our team has developed a novel biochar-based laboratory-scale polishing system that efficiently removes antibiotics from reclaimed water. Biochars are organic matter burnt in an oxygen-starved environment. They have high adsorption potential for organic compounds and can be used as a cost-effective material for filtration. Biochars prepared (engineered) from various feedstocks,pyrolysis temperatures, and post-pyrolysis modifications, have different antibiotic adsorption properties and physicochemical characteristics. Based on this knowledge, a biochar type can be designed for the specific antibiotic compounds found inreclaimed water.In the current study, we propose developing a large-scale, highly-effective antimicrobial resistance mitigation system that incorporates engineered biochar adsorbents tailored to specific contaminants in reclaimed water. We will assess the system's performance regarding removing ARDs from target reclaimed water. Moreover, we will use subsequent potted edible plants (e.g., spinach and radish) grown under greenhouse conditions to study the effect of reclaimed water on ARD dissemination and link treatment system efficacy to ecological parameters indicative of antimicrobial mitigation. Lastly, a pilot-scale system will be developed for coupling with a conventional field-scale crop irrigation system. The pilot biochar-based polishing system will be assessed for performance in reducing antimicrobial resistance dissemination in soils and plants irrigated with reclaimed water. Extension workshops for farmers and stakeholders will be conducted on the risks of antimicrobial resistance dissemination in agriculture and their mitigation with our biochar-based polishing systems. At workshops, we will demonstrate the biochar-based polishing system for reclaimed water and our findings on reducing antimicrobial resistance in soil and crops irrigated with polished reclaimed water. Risk assessments will also be conducted to quantify the extent to which the polishing system reduces human exposure to antimicrobial resistance via the food chain. The work will assist in curtailing antimicrobial resistance dissemination in agriculture to protecthuman health and the environment.
Animal Health Component
50%
Research Effort Categories
Basic
20%
Applied
50%
Developmental
30%
Goals / Objectives
Determine the efficacy of a broad spectrum of biochar materials for their ability to remove antibiotics, ARGs, and ARB from TMW and use the most effective materials.Design a bench-scale polishing system to treat synthetic and real TMWs using the biochars selected under Objective 1, comparing the levels of antibiotics, ARGs, and ARB in the influent and effluent to determine the removal efficacy of the system.Upscale and refine the polishing system, using influent and effluent solutions from the bench-scale polishing system to irrigate different crops under greenhouse conditions, comparing temporal changes in the microbial communities (including ARGs and ARB) of soils and plants irrigated with non-polished and polished TMW.Design, fabricate, and operate a scaled-up, on-farm biochar-based system that is coupled with an on-farm irrigation system to provide polished TMW for crop production under field conditions, comparing spatio-temporal changes in the microbial communities (including ARGs and ARB) of soils and plants irrigated with non-polished and polished TMWs; conduct field evaluations at two farms over two growing seasons.Quantify the risk mitigation potential offered by the biochar-based polishing system in terms of antimicrobial dissemination in the agriculture continuum.
Project Methods
Study design: Focusing on agricultural systems, this work will aim to mitigate antibiotic resistance in irrigated agriculture by using multilayered biochar-based technologies to polish TMWs prior to irrigation. As biochar has been previously shown to effectively adsorb a variety of contaminants, a broad spectrum of biochar materials (e.g., from various feedstocks and production conditions) will be assessed in terms of ARD removal from TMWs. The most effective biochar materials will be used to design an intermediate-scale polishing system that will be used for irrigating plants with polished TMWs. Several above- and below-ground vegetable types will be assessed, focusing on those eaten raw or with little processing. The work will be conducted using greenhouse and outdoor field experiments, which are of a sufficiently large scale to allow for realistic biological processes to take place while still being highly controllable. In the pot and field studies, we will assess changes in soil microbial composition as well as in the concentrations of ARDs in the soil, soil solution, rhizosphere, and phyllosphere in response to irrigation with both as-collected and polished TMWs. State-of-the-art techniques such as liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS, Q-Exactive Hybrid Quadrupole-Orbitrap), triple quadrupole LC-MS/MS, high throughput quantitative polymerase chain reaction (HT-qPCR), 16S rRNA gene amplicon sequencing, and metagenomic sequencing will be used throughout to produce high-quality data on the development and transfers of antibiotic resistance within the systems. The data obtained in this project will serve as the basis for quantitative risk mitigation of biochar-based polishing systems in terms of the dissemination of antibiotic resistance through food chains; thereby helping address global concerns about the potential for human exposure.Techniques:Biochar Characterization:These methods are collectively used to determine intrinsic and extrinsic characteristics and reactivity of produced biochar materials, essential for identifying structure-reactivity relationships between biochars and ARDs.Surface functional group composition will be probed by Fourier transform infrared (FTIR) spectroscopy. Biochar materials will be analyzed directly by FTIR on a Bruker Invenio S spectrometer and require no sample preparation beyond initial biochar grinding, which is necessary for all analyses we propose.Biochar carbon, nitrogen, oxygen, and hydrogen concentrations, which serve as important metrics for biochar polarity and hydrophobicity, will be determined through sample combustion (carbon and nitrogen) or pyrolysis (oxygen and hydrogen) on an Elementar Pyrocube.Moisture, volatile, ash, and fixed carbon contents of biochars will be determined using ASTM method D1762-84.Biochar porosity and specific surface area will be determined through N2 adsorption onto biochars.Biochar particle morphology will be probed by scanning electron microscopy (SEM).Surface charges for biochar particles will be measured with a Zeta-Meter 3.0 instrument.The cation exchange capacity (CEC) and pH of biochar materials will be determined using standard wet-chemical laboratory analyses.LC-(HR) MS/MS:For the detection of antibiotics in the as-collected TMWs, the influent, effluent, and interstitial solution of the biochar-based polishing systems, soils, and plant materials, LC-triple quadrupole MS/MS will be used.Analytical methods will be developed for the compounds of interest in a given treated wastewater. Detection of potential antibiotic transformation products formed during the TMW polishing process and after the irrigation will be examined using LC-high-resolution tandem mass spectrometry (LC-HRMS/MS).Co-PI Men's laboratory has established suspect screening and non-target screening workflows, which have been applied to identify and confirm the occurrence of emerging organic contaminants, as well as their transformation products in various matrices.Bacterial Community Analysis:The DNA extracted from treated municipal wastewater, water, biochar, plant surface, and soil will be assessed for quantity and quality on a Qubit.For bacterial community profiling, the V4 hypervariable region of the 16rRNA gene will be sequenced on an Illumina MiSeq platform.The reads for 16Sr RNA gene amplicons will be denoised and analyzed with DADA2, Phyloseq.The reads will be quality trimmed, denoised, and then run through the core DADA2 error correction algorithm. The resulting reads will be amplicon sequence variants (ASVs), chimera free that are analogous to OTUs.The ASVs will be assigned taxonomic identities using the SILVA 16S database.Following rarefaction, alpha diversity, and beta diversity analyses will be employed.Analysis of Similarities and non-metric dimensional scaling plots of distances will be used to compare treatments. Community resistance will be determined.Analysis of ARBs, ARGs, and MGEs:Cultivation and minimum inhibitory concentration assays for ARBs:To estimate the viable bacterial counts, treated municipal wastewater, water (influents and effluents), biochar, and soil sample suspensions will be serially diluted and plated on Mueller Hinton agar. Subsequently, no dilution fraction of the suspensions will be plated on a) MH agar with four antibiotics and b) Brilliance ESBL agar for isolation of viable ESBL-producing bacterial strains.A susceptibility testing will be performed for isolated MDR, and ESBL strains with Sensititre™Complete Automated AST System. After the susceptibility test, the strains that produce ESBL will be subjected to whole genome sequencing and genome extraction.Quantifying ARGs and MGEs:High-throughput quantitative PCR (HT-qPCR) with Takara SmartChip real-time PCR system will be used for the quantification of a) 283 ARGs, b) MGEs (eight transposons and four integron-integrase genes), and c) 16SrRNA gene. The relative copy numbers will be calculated for each gene.Droplet digital PCR will be used for absolute quantification of a) ARGs and b) MGEs, and c)16SrRNA. For each sample, eighteen target genes encoding resistance to the main antibiotic families in soil and wastewater, including sulfonamides (sul1 and sul2), tetracyclines (tetC, tetG, tetH, tetM, tetO, tetQ, tetS, tetW, tetB/P, tetT, and tetX), and chloramphenicol (fexA, fexB, cmlA, cfr and floR) will be quantified. In addition, two MGEs, integrase genes of class 1 (int1) and 2 (int2) integron and 16SrRNA gene, will also be quantified for each sample.Genome-based ecology of bacteria and their resistomes: Complete genomes of bacteria and their antibiotic resistomes will be extracted. High-quality DNA extracts will be sequenced on the Illumina NextSeq500 DNA sequencer. The raw reads will be quality filtered and de novo assembled into scaffolds. The assembled scaffolds will be binned with MetaBAT2. The binned scaffold quality will be assessed with CheckM. The bins will be assigned as metagenome-assembled genomes (MAGs). The gene prediction of MAGs will be performed with Prokka. The predicted genes will be annotated for function. To identify ARGs among the MAGs, we will use the resistance gene identifier of the CARD. The annotations of the MAGs will assist in the metabolic reconstruction of the bacteria. The ARG profiles (type and relative abundance, Bray-Curtis) will be compared using ANOSIM and NMDS plots. In addition, long-read metagenome sequencing with Pacific Biosciences RS11 DNA sequencer will be performed for samples with ARB MAGs.Acronyms: ARD: Antibiotic resistance determinants (Antibiotics, ARGs, ARBs, and MGEs); ARGs: Antibiotic resistance genes; ARB: Antibiotic-resistant bacteria; MGE: Mobile genetics elementsRefer to methods in Projective Narrative for the list of references.