Multi-Trophic Effects of Agricultural Microplastics: Implications for Soil Biological Health

MULTI-TROPHIC EFFECTS OF AGRICULTURAL MICROPLASTICS: IMPLICATIONS FOR SOIL BIOLOGICAL HEALTH

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

ACTIVE

Funding Source

AFRI COMPETITIVE GRANT

Reporting Frequency

Annual

Accession No.

1028529

Grant No.

2022-67019-37201

Cumulative Award Amt.

$750,000.00

Proposal No.

2021-09113

Multistate No.

(N/A)

Project Start Date

Jul 1, 2022

Project End Date

Jun 30, 2026

Grant Year

2022

Program Code

[A1401]- Foundational Program: Soil Health

Recipient Organization
UNIVERSITY OF TENNESSEE
2621 MORGAN CIR
KNOXVILLE,TN 37996-4540

Performing Department
Biosys Eng & Soil Science

Non Technical Summary
Plastic waste has inundated ecosystems around the world, and soils are particularly threatened by this seemingly ubiquitous pollution. Plastic film mulches used in many cropping systems are contributing to soil plastic pollution: most are made of polyethylene (PE), which is poorly degradable, and leaves fragments in soils for long periods. Biodegradable plastic mulch films (BDMs) are an alternative, meant to be tilled into soil after use and degraded by microorganisms. Plastic film remnants break down into microscopic pieces, or microplastics, which are accumulating in agricultural soils. Evidence continues to build that soil biota interact with microplastics with detrimental effects. However, the limited research to date has focused on conventional (i.e. non-biodegradable) plastics; impacts of biodegradable microplastics are unknown. Although microplastics derived from biodegradable plastic films will ultimately mineralize, they still reside in soil for several weeks to months. Thus, soil biota are exposed to biodegradable microplastics in potentially high concentrations, especially following till-down of BDMs. Our overarching goal is to reveal impacts of both conventional and biodegradable microplastics and nanoplastics on soil biota and their functions to better assess how plastic pollution affects soil health. Our driving questions are: 1) How do microplastics affect microbial and microfaunal communities and their functionality?; and 2) Do these impacts differ between conventional PE films and biodegradable plastic films? Our proposed research will use agriculturally-relevant microplastic fragments to investigate effects of PE and BDM microplastics on soil organisms, populations and communities in terms of physiology, diversity, structure, and their contributions to critical soil functions.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
133	0110	1060	100%

Knowledge Area
133 - Pollution Prevention and Mitigation;

Subject Of Investigation
0110 - Soil;

Field Of Science
1060 - Biology (whole systems);

Keywords

Goals / Objectives
Our overarching goal is to reveal impacts of both conventional and biodegradable microplastics and nanoplastics on soil biota and their functions to better assess how plastic pollution affects soil health. Our driving questions are: 1) How do microplastics affect microbial and microfaunal communities and their functionality?; and 2) Do these impacts differ between conventional polyethylene (PE) films and biodegradable plastic films (BDMs)? Our proposed research will use agriculturally-relevant microplastic fragments to investigate effects of PE and BDM microplastics on soil organisms, populations and communities in terms of physiology, diversity, structure, and their contributions to critical soil functions.

Project Methods
Objective 1: Organisms and PopulationsTask 1.1. Produce environmentally realistic micro- and nanoplastics from PE and BDM filmsWe will use commercially available mulch films as the starting material, in particular, a conventional PE film and a BDM: BioAgri™ (Biobag Americas, Dunedin, FL) (Hayes et al. 2019; Hayes et al. 2017). Plastic mulches will be cryogenically soaked to simulate the embrittlement that occurs during environmental weathering (not needed when starting materials are weathered), followed by mechanical milling to simulate the plowing of BDMs into soil (Astner et al. 2019 and Astner et al. in prep).Task 1.2 Soil bacterial population studiesWe will investigate whether exposure to 3 sizes of PE and BDM micro- and nanoplastics (0.3, 106 and 250 µm) influences microbial viability and functioning using a standard luminescent bacteria test for toxicity (ASTM D5660). After growth curve analysis to determine optimal media and timing for each strain, we will expose mid-log phase cultures of common soil bacteria to PE and BDM micro- and nanoplastics, along with no-plastic controls, and monitor responses after 1, 4 and 24 h exposure. Responses measured will include: 1) cell viability via colony counts on agar plates; 2) respiration, using CO2 evolution in culture bottle headspaces measured on an IRGA, as described in Bonifer et al. (2019); 3) general extracellular enzyme activity, measured using fluorescein diacetate (FDA)-ase activity (Green et al. 2006); 4) inorganic nitrogen (NH4 and NO3) uptake from media using standard methods; 5) oxidative stress via a fluorescence-based measurement of intracellular reactive oxygen species (Sun et al. 2018); 6) morphological changes. We will select the strains and plastic size/concentrations for further investigation using untargeted metabolomics and lipidomics using established methods at the UT Biological and Small Molecule Mass Spectrometry Core Lab (BSMMSC).Task 1.3. Soil faunaMillipedes (Diplopoda): We will use two millipede species (Oxidus gracilis, Narceus americanus), now in culture by co-PI Bernard. Microplastics will be applied to lettuce, thin cucumber slices or fresh corn kernels and weighed, then placed into millipede rearing containers. Periodically, millipedes will be dissected to determine commensal nematode loads and shifts in individual nematode taxa and life stages (Phillips et al. 2019). Presence of plastic particles in both millipedes and nematodes will be determined with microscopy and Fourier Transformed Infrared Spectroscopy (FTIR). Millipede frass (droppings) will be dispersed in water to determine the degree of physical breakdown of plastic particles during the digestive process using methods described in Task 1.4. We will remove intestines from the millipede body (Phillips et al. 2019) and section the middle third of the hindgut for analysis. DNA will be extracted from the hindgut contents for 16S rRNA (V4 region) library preparation and Illumina MiSeq sequencing as described in Task 2.3.Nematodes: We will determine effects of microplastics on more typical bacteria-feeding soil nematode species and assemblages, using three true soil nematode lineages: Rhabditidae (Rhabditella axei or Distolabrellus veechi, extreme c-p1 nematodes), Diplogastridae (Pristionchus pacificus (c-p1) and Cephalobidae (Acrobeloides nannus (c-p2)). These species will be stock-cultured on NGM agar streaked with E. coli OP-50 (Stiernagle 1999). NGM agar with OP-50 will be surface-coated with microplastic particles; developmental rates, reproduction and survival in both single and multiple-species cultures will be monitored.Springtails (Collembola): We will conduct competition studies to estimate the differential effects of microplastics on three springtail species with different field microhabitats: euedaphic (Protaphorura fimata, a known plant pest); hemiedaphic (Folsomia candida); and surface litter (Sinella curviseta), and observe changes in development rates, fecundity and survival in the presence of microplastics of a range of sizes.Task 1.4. Material characterization of microplasticsThe size distribution of microplastics will be determined with stereomicroscopy, followed by Image J analysis. We will determine the extent of depolymerization of microplastics and the molecular weight distribution through gel permeation chromatographic analysis and thermogravimetric analysis (TGA). NMR will be used to quantify the relative proportion of PBAT monomeric units in BDM particles (Hayes et al. 2017; Herrera et al. 2002).Objective 2: CommunitiesTask 2.1 Conduct greenhouse scale pot studySoil will be divided into 7 treatment groups: two types of microplastics (PE and BioAgri BDM) at three concentrations (1%, 5%, and 10% w/w) plus a no plastic control. Each treatment combination will have three replicate pots. Microplastics will be generated (Task 1.1), thoroughly mixed with the soil and divided into 3.8 L (1 gallon) pots. Pots will be planted with a mixture of three or more seedlings of diverse phylogeny (e.g., clover, fescue, brassica) and kept in the greenhouse under controlled temperature, humidity, and lighting conditions. Pots will be destructively sampled after 0.5, 1, 2, 4, 8 and 12 months to determine short and long-term changes in nematode and microbial communities (Tasks 2.2-2.4).Task 2.2 Characterize nematode communities in pot studiesCommunities will be analyzed for shifts in species composition, trophic levels and colonizer-persister (c-p) functional groups (Bongers & Bongers 1998) as we have previously done (Keenan et al. 2018; Taylor et al. 2020).Task 2.3 Structure of microbial communities in pot studiesSoils collected from the greenhouse pot study will be characterized for bacterial and fungal communities via 16S and ITS amplicon sequencing as we have done previously (Ashworth et al. 2017).Task 2.4. Biogeochemical function of microbial communities in pot studiesTwo main carbon pools will be measured: microbial biomass carbon (MBC) and soil organic carbon (SOC). As a metric of general microbial activity, we will measure fluorescein diacetate hydrolytic, FDAase (Green et al. 2006). We will also measure potential activity of specific C and N cycling extracellular enzymes including β-glucosidase, cellobiohydrolase, and N-acetylglucosaminidase (Sinsabaugh et al. 2008). In addition, we will employ untargeted metabolomics to profile the suite of small water-soluble metabolites being produced by the communities as described for Task 1.2.Objective 3: Tracing Plastic Carbon in SoilTask 3.1. Isotopic probing of microplastic impacts on soil carbon cyclingAs a first study to gain fundamental understanding on how plastic-C cycles in soil in comparison with SOC, we will use a stable C (13C) isotope labelling approach. We will set up an incubation experiment for six months using the same soil used for the greenhouse study (Task 2.1). We will use poly(ethylene-13C2) (>99% 13C2-labelled powder, Sigma-Aldrich) to represent a conventional plastic mulch and oligo or poly(lactic acid-13C2), synthesized in Co-PI Hayes's lab via lipase-catalyzed polycondensation (Hayes et al. 2012; Kelly & Hayes 2006) or catalytically (Lim et al. 2010; Mehta et al. 2005) using >99% 13C2-labelled L-Lactic acid-1-13C powder (Sigma-Aldrich), as a representative BDM polymeric material (Hayes et al. 2017). Total CO2 and 13CO2 concentrations in headspace samples will be measured at the UC Davis Stable Isotope Facility.Task 3.2. Determine microbial transformation of microplastics and/or impacts on carbon cycling functions using stable-isotope enabled metabolomicsTo determine transformation products of microplastic degradation and reveal potential microbial catabolic pathways involved, we will use stable isotope-enabled metabolomics, as described in Mosier et al. (2013) and Tian et al. (2018).

Progress 07/01/24 to 06/30/25

Outputs
Target Audience:Our research on the effects of soil microplastics on soil biota has been shared with scientists, USDA personnel, graduate students, research associates, environmental professionals, and K12 educators. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Undergraduate student training: Two summer undergraduate students conducted independent research projects related to soil microplastics: One student (NSF REU Site Program) tested different methods for sterilization of plastics that could be used in microbiological research. One student(UT-Oak Ridge Innovation Institute SMART Internship Program) conducted a study analyzing metabolomic changes in microbes under combined exposure of microplastics and PFAS. Graduate student training:The PhD student presented her research at ASM Microbe in Jun 2025. She was additionally selected to participate the American Society for Microbiology Future Leaders Mentorship Fellowship (FLMF), a 2-year program that matches master's and doctoral students in the microbial sciences with experts (mentors) across varied career sectors. This program will facilitate mutual leadership growth and learning, help participants build their mentoring portfolios, provide networking opportunities with ASM members and non-students alike and increase social capital between fellows and mentors.TheMS studentparticipated in the collection of ultra-small-angle neutron scattering at Oak Ridge National Laboratory in May 2025. How have the results been disseminated to communities of interest?SMART Symposium (07/2024): Summer undergraduate student, mentored by PhD student, presented her summer research on agricultural impact of multi- contaminant exposures on bioplastic degrading Bacillus pumilus B12 metabolomes to an audience with mixed scientific expertise. Department of Microbiology Annual Retreat (08/2024): Summer undergraduate student, mentored by graduate student, presented his summer research project on microbial growth on bioplastics to an audience of microbiologists. Frontiers in Biorefining Conference (09/2024): Graduate studentpresented results on the metabolic response of Bacillus to plastic exposure to an audience of environmental chemists. Backyard STEM (12/6/2024): DeBruyn co-leads Backyard STEM, a curriculum development and trainer program for Tennessee 4-H educators. Each year, new activities are developed and disseminated through regional training inservices. This year, curriculum focused on the UN sustainable development goals and highlighted how renewable materials such as biodegradable plastics are a part of sustainable development strategies. Around 50 agents across Tennessee were trained on the new material and will use the materials in their upcoming 4-H programming. AAAS Symposium (02/2025): Hayes and DeBruyn pitched a symposium focused on agricultural microplastics for the American Association for the Advancement of Science general meeting and it was accepted for February 2025. Presenters were invited from Cal Poly and Washington State University, and approximately 100 people attended. ASM Microbe (06/2025): Graduate student presented results on the influence of microplastics on soil microbial communities to other microbiologists. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Determine the concentration-dependent effects of BDM and PE microplastics and nanoplastics on individual soil organisms and populations. To determine the shapes and sizes of the microplastic feedstock fragments we continue to generate and are using across the experiments, we will continue characterize the microplastics using microscopy and image analysis via Image J and determine median sizes of the fragments. To explore the effects of microplastics on soil bacteria, we will extend the methods we used for Pseudomonas monteilii to additional strains in our culture collection. Specifically, we plan to test two rhizosphere isolates from switchgrass: the Gram-negative Ralstonia pickettii and the Gram-positive Priestia megaterium, for which we have already determined growth kinetics to enable us to select the appropriate time points for testing. These strains were chosen to represent phylogenetically and physiologically distinct groups of soil bacteria with different cell wall structures. By examining physiological responses such as growth dynamics, intracellular metabolite profiles, and lipid production under microplastic exposure, we aim to determine whether the effects observed in P. monteilii are strain-specific or broadly applicable across diverse bacterial taxa. The inclusion of both Gram-negative and Gram-positive species will also allow us to assess whether differences in cell envelope architecture play a role in mediating bacterial responses to microplastic stress, thereby helping us understand whether observed impacts are taxonomically constrained or represent a more generalizable microbial response. In a previous experiment a cohort of millipedes fed their entire lives on cucurbit slices (cucumber, squash). A new experiment was developed to determine the effects of a natural diet on the microbiome of millipedes living in nature. Thirty adult N. americanus were collected from a local natural area. Ten were immediately dissected and their gut contents collected as described above. Two sets of ten each have been prepared and are being fed cucurbits. Each set of 10 will be subdivided into two subsets of five, with one subset receiving PBAT-coated food and the other subset serving as a control. After one month one set of 10 will be dissected and the gut contents analyzed as before; after four months the other set will be analyzed. We will compare the microbiomes of the wild populations and lab reared populations to reveal differences in microbiome and if the effects of plastic feeding differ for wild and lab-reared millipedes. Effects of microplastics on springtails (Collembola) will continue with other species in addition to Yuukianura aphoruroides, based on their ease of culture, close relation to soil and litter environments, and manipulability (Folsomia candida, Protaphorura fimata, Sinella curviseta). We will transfer adult specimens into small containers containing a clay substrate. These containers will be maintained in darkness and provided with food twice a week, with regular recording of egg production until all containers are actively producing. To assess fecundity for P. fimata and S. curviseta (obligate mating), we will pair the containers based on reproductive output, creating five pairs in total. One set of five pairs will serve as the control group, while the other set of five pairs will constitute the treatment group. Folsomia candida is parthenogenetic. The control group will receive a diet from Springtails US, while the treatment group will be fed a diet comprising 20% microplastics. To prepare the treatment group's diet, microplastics will be blended with water in a weigh boat to create a slurry, which will then be air-dried overnight and flaked to form the diet. We will count laid eggs and adult population within each container. In case of adult mortality, dissections will be conducted to determine the presence of microplastics in the digestive system. This experiment will continue until a significant change occurs in fecundity, until the springtails exhibit adverse effects from the microplastic exposure, or the lack of an effect is clear. Nematode experiments will continue with tests on soybean cyst nematode (Heterodera glycines) similar to those reported above for Meloidogyne incognita. In addition, three free-living bacterivorous nematodes now in agar culture will be examined for reaction to microplastics. These nematodes are Distolabrellus veechi, Pelodera sp., and an unidentified rhabditid nematode. Two separate experiments will be performed: MPs sprinkled on the surface of NGM agar and a three-dimensional experiment consisting of fine sand mixed with MP particles embedded in dried agar, which then will be moistened in situ and nematodes added to the moistened sand. In both cases, treatments will be evaluated for population dynamics by extraction and counting of nematodes. Objective 2. Determine the effects of BDM and PE microplastic loading levels on soil community structure and function. Untargeted metabolomics was used to determine how microplastics affected soil biological functional (metabolic) responses. Currently, we are in the stage of analyzing the unknown metabolite features, while also working on the overall dataset. Since all samples were run in a randomized design, we plan to apply SERF normalization to account for technical variability and improve comparability across treatments. This stage will provide a comprehensive view of both known and novel metabolic responses of soil microbial communities to microplastic exposure. We will finish our comparison of DNA sequencing approaches to microscopy for characterizing nematode communities. Our preliminary results suggest that DNA approaches may underestimate nematode diversity in samples based on the relative differences in Rhabditida and Dorylaimida reported. This may be attributed to the differences in soil volumes analyzed: extraction methods for microscopy require 100 g soil, whereas DNA extraction the soil requirement is 250 mg. Further, we are noting that sequencing approaches are limited due to poor taxonomic identification associated with sequences that are found in the Silva database and across Phylum Nematoda. Recently, a newly curated database of nematode sequences has been produced (NemaTaxa) designed to be compatible with both Mothur and QIIME analysis platforms (Baker et al. 2023). The authors corroborated our findings that the Silva 132 release only supports classification to order level. The NemaTaxa database is composed of sequences downloaded from NCBI and aligned to the primers NF1 and 18Sr2b, with incomplete lineages corrected. We plan to align our sequences to this database next and then compare these results to our microscopy data. Biological (nematodes and microbes) and soil chemical data will be combined, and multivariate analysis will be used to identify concentration-dependent drivers of community change due to exposure to microplastics. A manuscript describing the findings from this experiment will be prepared and submitted to mSystems (or similar journal). Objective 3. Determine the effects of BDM and PE microplastics on microbial C cycling. The experiment and the sample analysis are complete, and manuscript revisions have been submitted to Journal of Environmental Quality.

Impacts
What was accomplished under these goals? Objective 1: Determine effects of BDM and PE microplastics on soil organisms. Our project seeks to determine the impacts of agricultural microplastics on soil biota. Agriculturally relevant microplastics were produced from both conventional (PE) and biodegradable (PBAT) films using a combination of grinding and sieving to achieve a desired size range. We produced approximately 2 kg of microplastics with a size of <250 µm: particle size analysis revealed that 90% of the particles were between 100 and 250 µm. These have been fully characterized for size and molecular composition and were used across several ongoing experiments. To determine effects on soil microbes, we have focused on Pseudomonas monteilli, a Gram-negative soil bacterium. P. monteilli was exposed to low-density polyethylene (LDPE) and polybutylene adipate terephthalate (PBAT) microplastics at a concentration of 0.02% w/v. We evaluated the impact of microplastics on the organism's growth, lipid profile, and metabolite expression at four different time points during its growth. Growth was assessed using live/dead fluorescence staining, while lipidomic and metabolomic profiles were analyzed using ultra-high-performance liquid chromatography (UHPLC). The results suggest no significant differences in overall growth between treatments with LDPE and PBAT, regardless of the growth phase and lipidomic profiles did not show clear distinctions between the two plastic types. Metabolomic data analysis is ongoing. To determine effects on millipedes, mid-size juveniles of the American Giant Millipede, Narceus americanus, were given either PBAT discs sandwiched between two cucumber slices, ground PBAT (25 mm3, particle diameter <250 µm) sprinkled between the slices, or cucumber only. Although these millipedes exhibited individualistic feeding behaviors, most ate through the plastics. Microplastic particles were observed in fecal pellets confirming consumption. A separate choice experiment was conducted: In general, cucumber only was consumed before the microplastic-contaminated cucumber, suggesting millipedes may avoid food contaminated with microplastics. To determine effects of microplastic ingestion on the gut microbiome of the millipede Narceus americanus, we collected fecal pellets throughout the aforementioned experiment. Additionally, millipedes were dissected, the intestine was removed and opened, contents of the posterior gut were scraped out with a sterile microspatula. DNA was extracted from the pooled fecal pellets and the gut contents, and amplicon sequencing of 16S rRNA genes was used to characterize the microbial communities (Data analysis is ongoing). To test a second and much smaller species of millipedes, mated adult pairs of Oxidus gracilis were fed cucumber slices sprinkled with LDPE or PBAT (diam. <250 µm). Reproduction as measured by the appearance of newly hatched juveniles was not affected by any treatment. PBAT was ingested by adult O. gracilis but LDPE was not; instead, LDPE particles were scattered on the substrate surface caused by millipede movement over the cucumber.The springtail Yuukianura aphoruroides was fed PBAT + yeast. The springtail ate the yeast leaving behind the PBAT. Survival and egg production were not affected compared to the controls. Specimens did not appear to ingest PBAT as determined by dissection. Objective 2. Determine the effects of BDM and PE microplastic loading on soil community structure and function. To determine the effects of microplastics on soil communities, we conducted a 6-month long greenhouse study with field soils mixed with 0.2%, 1% and 2% w/w concentrations of PE and PBAT microplastics. To determine the effects on nematode communities, nematodes were extracted from 100 g soil using sugar-flotation centrifugation and identified to primarily genus level by microscopy. Overall, abundance, richness, and alpha (Shannon) diversity were not significantly affected over six months of exposure to either type of microplastics. When nematodes were sorted according to feeding groups (bacterivores, fungivores, plant associates, omnivores, and predators), an increase in bacterivores was observed in soils containing LDPE, whereas fungivores increased in soils containing PBAT. In addition to microscopic identification, we also explored the use of high throughput sequencing for characterizing nematode communities. Amplicon libraries were made using the nematode-specific primers NF1 and 18Sr2b and sequenced on the Illumina NovaSeq platform. Sequences classifying as phylum Nematoda were selected for analysis, generating a count table consisting of 1,230 taxa. Sequencing methods yielded 12 taxa across both family and order taxonomic ranks. In comparison, microscopy methods had yielded 41 uniquely identifiable live taxa across 26 families and 8 orders. While both microscopy and sequencing techniques yielded eight orders, only five of these overlapped: Chromadorida, Dorylaimida, Monhysterida, Rhabditida, Triplonchida. Rhabditida dominated sample composition in microscopy samples, however Dorylaimida frequently dominated the sequencing libraries. Bacterial and fungal communities - 16S and ITS amplicon libraries indicates that PBAT microplastics at high concentration led to a reduction in richness and diversity relative to controls, suggesting that the presence of microplastics might negatively impact microbial communities. Also, there is transient decrease in bacterial diversity at T2 (2 weeks) to T5 (4 months), particularly in the high PBAT treatments. However, these effects were no longer apparent by T6 (6 months), suggesting that the degradation process may have plateaued. PBAT also resulted in a stronger change in fungal communities relative to PE. In contrast to bacterial communities, the fungal communities became more divergent over the course of the experiment. Untargeted metabolomics was employed to investigate how microplastics affect soil biological functions. Soil samples were solvent-extracted and analyzed via LC/MS-MS at the UT Biological Small Molecule Mass Spectrometry Core Lab (BSMMSC). Following data acquisition, raw files were converted to centroid mode using MS-Convert to enable downstream processing. Known metabolites were then annotated and quantified using El-MAVEN with reference libraries. Data analysis is ongoing. Objective 3. Determine the effects of BDM and PE microplastics on microbial C cycling. To determine if microbial degradation of biodegradable plastics, which are intentionally tilled into soil, may accelerate or inhibit the mineralization of native SOC, we used a 193-day incubation experiment to investigate the degradation and priming effects of three biodegradable microplastics (polybutylene succinate (PBS), polylactic acid (PLA), and a PLA-polyhydroxyalkanoate blend (PLA/PHA)) compared to a conventional microplastic (low-density polyethylene (LDPE)), in agricultural soil under low and high N conditions. Isotope (13C) tracing allowed us to determine the cumulative loss of plastic- vs. SOC-derived carbon (C) as CO2-C. Biodegradable microplastics varied in biodegradation rates and priming effects, with PLA/PHA losing the most plastic-C (17.88%) and inducing the greatest positive priming effects (371 μg C g dry soil-1). In contrast, PLA, PBS, and LDPE showed <1% plastic-C loss and weaker priming effects ranging from positive to negative (73.1, 8.81, and -45.4 μg C g dry soil-1, respectively). Although N addition decreased the total C loss from both native SOC and microplastics, it did not alter priming effects. We conclude that biodegradable microplastics may threaten native SOC pools, and higher N availability may promote persistence of biodegradable plastics in soils.

Publications

Type: Peer Reviewed Journal Articles Status: Under Review Year Published: 2025 Citation: Wooliver R, A Neupane, JM DeBruyn, DG Hayes, A Astner, S Jagadamma Biodegradable microplastics contribute to soil organic carbon loss from agricultural soil. Journal of Environmental Quality. Resubmitted after revision
Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Ajide-Bamigboye TN, N McCants, JM DeBruyn, AL May. Agricultural impact of multi-contaminant exposures on bioplastic degrading bacteria Bacillus pumilus B12 metabolomes. Frontiers in Biorefining Conference, St. Simons Island, GA, 30 Sept 3 Oct, 2024
Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Ajide-Bamigboye TN, LS Taylor, A Astner, A Ajibola, DG Hayes, JM DeBruyn. Influence of plastic mulch film microplastics on soil microbial communities. American Society for Microbiology, Los Angeles, CA, 19-23 Jun 2025
Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: DG Hayes (moderator), M Flury, SM Schaeffer, S Sistla, 2025, Do Microplastics in Agricultural Ecosystems Threaten Food Supply? (Symposium) AAAS Conference, Boston, 14 February

Progress 07/01/23 to 06/30/24

Outputs
Target Audience:Scientists, NRCS and USDA personnel, graduate students, research associates, environmental professionals, educators. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Bioinformatics training 1: Nimat Ajide-Bamigboye (PhD student) completed the Mothur workshop, a training course devoted to bioinformatic and statistical analysis of community sequencing data (Spring 2024) Bioinformatics training 2: Lois Taylor (post doc) attended a workshop at the Society of Nematologists' annual meeting that focused on optimizing bioinformatics pipelines and analysis strategies for nematode sequencing data (August 2024). Undergraduate student training: Two summer interns (Summer 2024) conducted independent research projects related to microplastics. One project focused on sterilization approaches for microplastic research, the other focused on metabolic response by bacteria to exposures of mixtures of microplastics and PFAS. Their work was presented at end-of-program symposia in July/August 2024. How have the results been disseminated to communities of interest? Microbial Insights Webinar (5/14/2024): DeBruyn gave a webinar for environmental professionals entitled: Are Biodegradables a Solution to Agriculture's Plastic Problem? hosted by Microbial Insights, a biotechnology company that uses molecular microbial tools to assess bioremediation and other environmental microbiology processes. Approximately 300 professionals attended the live webinar, and it is currently available through Microbial Insights as an On-Demand class. Microbiology Departmental Colloquium (11/2/2023): PhD student Nimat Ajide-Bamigboye presented her research entitled "Microplastics in the soil: effects on growth of soil microorganisms" to an audience of approximately 100 microbiologists. Backyard STEM (12/6/2023): DeBruyn co-leads Backyard STEM, a curriculum development and trainer program for Tennessee 4-H educators. Each year, new activities are developed and disseminated through regional training inservices. This year, curriculum focused on sustainable practices in our own backyards, including reducing our use of plastics. Around 50 agents across Tennessee were trained on the new material, and will use the materials in their upcoming 4-H programming. USDA Soil Health (A1401) Project Director Meeting (04/10/2024): DeBruyn presented an update on this project at the annual project director's meeting in Kansas City, MO. Approximately 50 other project directors funded under the Soil Health program were in attendance. Conference presentation (06/12/2024): DeBruyn JM, LS Taylor°, N Ajide-Bamigboye°, A Astner, A Ajibola, A Neupane, DG Hayes, EC Bernard, S Jagadamma. Impacts of plastic mulch film microplastics on soil nematode communities. Soil Science Society of America Bouyoucos Summer Conference, San Juan, Puerto Rico, 10-12 Jun 2024 SMART Symposium (07/25/2024): Summer undergraduate student Na'lanie McCants presented a poster and oral presentation of her project: "Agricultural impact of multi-contaminant exposures on bioplastic degrading Bacillus pumilusB12 metabolomes" at the SMART Internship Program Symposium in Knoxville, TN. Departmental retreat presentations (08/02/2024): Summer undergraduate student Jadon Gonzales, presented a poster describing his summer research project: "Determination of microbial contaminants and testing of sterilization techniques on lab-generated microplastics" and PhD student Nimat also presented the nematode results from this project: "Impacts of plastic mulch film microplastics on soil nematode communities" at the Department of Microbiology Annual Retreat in Oak Ridge, TN.Approximately 100 people in attendance at this poster session. What do you plan to do during the next reporting period to accomplish the goals? Objective 1: Determine the concentration-dependent effects of BDM and PE microplastics and nanoplastics on individual soil organisms and populations. To determine the shapes and sizes of the microplastic feedstock fragments we continue to generate and are using across the experiments, we will continue characterize the microplastics using microscopy and image analysis via Image J and determine median sizes of the fragments. To explore effects of microplastics on soil bacteria and fungi, we will continue to use the strains we now have in our culture collection and test individual strains for physiological responses to microplastic exposure. To determine effects of microplastics on soil nematode activities, we will conclude our experiment with root knot nematodes to determine if the microplastic treatments affected root penetration. To explore effects of microplastics on springtails (Collembola) we plan work with several species known for their ease of culture and manipulability (Folsomia candida, Protaphorura fimata, Sinella curviseta, and an undescribed species, "Neanura growae"). We will transfer adult specimens into small containers containing a 1:1 mixture of plaster of Paris and activated charcoal substrate. These containers will be maintained in darkness and provided with food twice a week, with regular recording of egg production until all containers are actively producing. To assess fecundity, we will pair the containers based on reproductive output, creating five pairs in total. One set of five pairs will serve as the control group, while the other set of five pairs will constitute the treatment group. The control group will receive a diet from Springtails US, while the treatment group will be fed a diet comprising 20% microplastics. To prepare the treatment group's diet, microplastics will be blended with water in a weigh boat to create a slurry, which will then be air-dried overnight and flaked to form the diet. We will count laid eggs and adult population within each container. In case of adult mortality, dissections will be conducted to determine the presence of microplastics in the digestive system. This experiment will continue until a significant change occurs in fecundity or until the springtails exhibit adverse effects from the microplastic exposure. To determine effect of microplastic ingestion on the millipede Narceus americanus, we will dissect the millipedes and evaluate the gut microbiome using amplicon sequencing. We will collect microplastic pieces from the millipede dung for material characterization to determine what changes they undergo within the gut. Objective 2. Determine the effects of BDM and PE microplastic loading levels on soil community structure and function. Nematode community analysis will be performed on sequencing data, and results will be compared with data generated microscopically. Untargeted metabolomics will be used determine how microplastics affected soil biological functional (metabolic) responses. Archived samples will be solvent-extracted and run on a LC/MS-MS at the UT Biological Small Molecule Mass Spectrometry Core Lab (BSMMSC). Peaks will be compared to the extensive libary of biological metabolites maintained by the BSMMSC and community metabolome profiles compared using multivariate statistical approaches (e.g. PLSDA). Where effects are seen, we will identify individual metabolites and pathways that are most differentially expressed between treatments in order to develop mechanistic hypotheses about the response of soil communities to microplastic exposure. Biological (nematodes and microbes) and soil chemical data will be combined, and multivariate analysis will be used to identify concentration-dependent drivers of community change due to exposure to microplastics. Objective 3. Determine the effects of BDM and PE microplastics on microbial C cycling. The experiment and the sample analysis are complete. Final data organization and analysis will be completed and manuscript will be prepared for publication.

Impacts
What was accomplished under these goals? Objective 1: Determine the concentration-dependent effects of BDM and PE microplastics and nanoplastics on individual soil organisms and populations. Our project seeks to determine the impacts of agricultural microplastics on soil biota. Most other studies use model microplastics (e.g. spheres), and we now know that size and morphology of microplastics are key determinants in their effects on biology. To that end, agriculturally-relevant microplastics were produced from both conventional (PE) and biodegradable (PBAT) films using a combination of grinding and sieving to achieve a desired size range. We produced approximately 2 kg of microplastics with a size of <250 µm: particle size analysis revealed that 90% of the particles were between 100 and 250 µm. These have been fully characterized for size and molecular composition and were used across several ongoing experiments. To determine effects of microplastics on important soil microbes, we established a collection of soil bacterial and fungal strains: Bacillus pumilus B12, Paraburkholderia, Variovorax, Pseudomonas chlororaphis, Rhizobium, Streptomyces, and a Trichoderma sp. fungus. To assess the impact of microplastics, we introduced PBAT and LDPE at a concentration of 0.2% w/v into R2A medium, using medium alone as a control. Focusing on B. pumilus B12, we examined the effects on growth and metabolism. Growth was assessed using plate counts, and metabolic changes were analyzed using mass spectrometry-based untargeted metabolomics. Preliminary findings indicate that at the end of the exponential phase (6 hours post-exposure), we observed an increase in growth compared to the control, while during the stationary phase (24 hours post-exposure), a decrease in growth was noted.Untargetedmetabolomics analysis revealed 115 metabolites across all time points. Notably, there were differences in the metabolite profiles produced in the presence of each type of microplastic compared to the control, suggesting microplastics may influence both the growth and metabolic activity of soil microbes in a time-dependent manner. To determine the effects of microplastics on soil nematodes, we established a test system with root knot nematode, Meloidogyne incognita. Cucumber (Cucumis sativus) seeds were sprouted and transplanted to 13-cm conetainers to maximize nematode interaction with plant roots. The experimental treatments were 0%, 0.2%, 1.0%, and 2.0% w/w microplastics (LDPE, PLA/PHA in separate experiments) mixed into an artificial substrate of fine sand, coarse sand, and Promix (2:1:1 ratio). After seedlings established, we introduced 500 nematode eggs into each tube. At the conclusion of the experiment, roots were harvested and stained to visualize nematodes that successfully colonized the roots.Regardless of microplastic density, root invasion was similar and not significantly different. To determine the effects of microplastics on millipedes, mid-size juveniles of the American Giant Millipede, Narceus americanus, were given 3 food sources: treatment 1 had a PBAT disc sandwiched between two cucumber slices, treatment 2 had ground PBAT (25 mm3, particle diameter <250 µm) sprinkled between the slices, and treatment 3 was a control with no microplastics. Although these millipedes exhibited individualistic feeding behaviors, most ate through the plastics.Microplastic particles were observed in fecal pellets confirming consumption. A separate choice experiment was conducted with control and sprinkle sandwiches in each container: In general, the control sandwich was consumed first before the sprinkle sandwich was fed upon, suggesting Millipedes may avoid food contaminated with microplastics. Objective 2. Determine the effects of BDM and PE microplastic loading levels on soil community structure and function. To determine the effects of microplastics on communities, we conducted a 6-month long greenhouse study with field soils and natural communities. We mixed in either PE and PBAT microplastics at 0.2%, 1% and 2% w/w concentrations. Control pots had no microplastics added. Pots were kept under relatively constant temperature and humidity in a greenhouse and destructively sampled at 1, 2, 5 weeks, 2, 4, and 6 months. Soil physicochemical parameters were measured, nematode communities were extracted and counted, and aliquots of soil were flash frozen and stored at -80°C for DNA- and metabolomic analyses. Elevated respiration, decreased soil organic carbon and nitrate, and elevated peptidase activity with the PBAT treatments indicated biodegradation was occurring. Nematode communities - Free-living soil nematodes are sensitive to environmental conditions, and fluctuations within their community structure often reflect changes to soil conditions. To determine the effects of microplastics on nematode community structure dynamics, we identified and quantified nematodes: Nematodes were extracted from 100 g soil using sugar-flotation centrifugation, and identified to primarily genus level by microscopy. Overall, abundance, richness, and alpha (Shannon) diversity were not significantly affected over a period of six months of exposure to either type of microplastics. When nematodes were sorted according to feeding groups (bacterivores, fungivores, plant associates, omnivores, and predators), an increase in bacterivores was observedin soils containing LDPE, whereas fungivores increased in soils containing PBAT. DNA was extracted from soils, sequenced using the nematode-specific primers NF1 and 18Sr2b on the Illumina NovaSeq platform and analyzed using a modified Mothur pipeline developed specifically for nematode sequences. Forthcoming analyses will include beta diversity (community change over time) and involve the comparison between data generated through microscopic techniques and sequencing to determine the relative strengths of both approaches. Bacterial and fungal communities - Sequencing of 16S and ITS amplicon libraries was performed at the UT Genomics Core on the Illumina NovaSeq platform. Bacterial sequences were processed using the Mothur pipeline, followed by analysis in R. Our analysis indicates that microplastics (LDPE and PBAT) led to a reduction in richness and diversity relative to controls, suggesting that the presence of microplastics might negatively impact microbial communities. The NMDS plot analysis suggests that different treatments have an impact on the microbial community structure. We will be focusing on the analysis of fungal communities next, applying similar sequencing and bioinformatic approaches to understand the impact of microplastics on fungal diversity and community structure. Objective 3. Determine the effects of BDM and PE microplastics on microbial C cycling. To determine effects of microplastics on soil carbon dynamics, a soil incubation experiment was conducted using PE and 3 biodegradable plastic feedstocks: polybutyl succinate (PBS), polylactic acid (PLA), and a polylactic acid/polyhydroxyalkanoate (PLA/PHA) blend. These were selected because their 13C signatures were distinct from each other and also distinct from background soil, allowing us to trace plastic-derived carbon to soil carbon pools. 1 g of microplastics (<500 um) were added to 50 g soil (2% w/w), along with a no plastic control. Because we have seen from previous work that nitrogen is a major driver for C cycling, we tested N addition as a second factor(ammonium nitrate at a rate equivalent of 125 kg/ha). Total and 13C-CO2 was measured at regular intervals throughout the experiment, and destructive samples were taken at 5, 10, 30, and 193 days to be analyzed for 13C, total C and N, microbial biomass carbon, mineral N, and pH.Preliminary data analysis shows that PLA/PHA biodegraded to a greater extent in soil compared to other BDMs, and resulting in priming of native SOC and immobilization of soil mineral N. The addition of N slowed the decomposition and priming effect.

Publications

Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: DeBruyn JM, LS Taylor, N Ajide-Bamigboye, A Astner, A Ajibola, A Neupane, DG Hayes, EC Bernard, S Jagadamma. Impacts of plastic mulch film microplastics on soil nematode communities. Soil Science Society of America Bouyoucos Summer Conference, San Juan, Puerto Rico, 10-12 Jun 2024

Progress 07/01/22 to 06/30/23

Outputs
Target Audience:Scientists, graduate students, research associates, environmental professionals, educators. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The postdoc working in the Jagadamma Lab has been training students on Carbon Isotope Gas Analyzer, which is heavily used in this project. Two new graduate students are working on this project and are learning skills in microplastic formation and manipulation in the lab. How have the results been disseminated to communities of interest? EnviroClass: DeBruyn conducted a training webinar for environmental professionals on Microplastics as an Emerging Contaminant through EnviroClass (www.enviroclass.com), a virtual professional development platform. Approximately 150 professionals attended the live webinar, and it is currently available through EnviroClass as an On-Demand class. European Bioplastics: DeBruyn presented on the effects of biodegradable mulches on soil health at the Roundtable on Soil Biodegradable Mulch Films hosted by European Bioplastics. Approximately 18 international scientists and policy makers were in attendance. Backyard STEM: DeBruyn co-leads Backyard STEM, a curriculum development and trainer program for Tennessee 4-H educators. Each year, new activities are developed and disseminated through regional training inservices. This year, curriculum focused on concepts around how human activities and pollution such as microplastics, interrupt natural cycles. Around 40 agents across Tennessee were trained on the new material, and will use the materials in their upcoming 4-H programming. What do you plan to do during the next reporting period to accomplish the goals? Objective 1: Determine the concentration-dependent effects of BDM and PE microplastics and nanoplastics on individual soil organisms and populations. To determine the shapes and sizes of the microplastic feedstock fragments we have generated and are using across the experiments, we will characterize the microplastics using microscopy and image analysis via Image J and determine median sizes of the fragments. To explore effects of microplastics on soil bacteria and fungi, we will use the strains we now have in our culture collection and test individual strains for physiological responses to microplastic exposure. To determine effects of microplastics on soil nematode activities, we will conclude our experiment with root knot nematodes to determine if the microplastic treatments affected root penetration. To explore effects of microplastics on springtails (Collembola) we plan work with several species known for their ease of culture and manipulability (Folsomia candida, Protaphorura fimata, Sinella curviseta, and an undescribed species, "Neanura growae"). We will transfer adult specimens into small containers containing a 1:1 mixture of plaster of Paris and activated charcoal substrate. These containers will be maintained in darkness and provided with food twice a week, with regular recording of egg production until all containers are actively producing. To assess fecundity, we will pair the containers based on reproductive output, creating five pairs in total. One set of five pairs will serve as the control group, while the other set of five pairs will constitute the treatment group. The control group will receive a diet from Springtails US, while the treatment group will be fed a diet comprising 20% microplastics. To prepare the treatment group's diet, microplastics will be blended with water in a weigh boat to create a slurry, which will then be air-dried overnight and flaked to form the diet. We will count laid eggs and adult population within each container. In case of adult mortality, dissections will be conducted to determine the presence of microplastics in the digestive system. This experiment will continue until a significant change occurs in fecundity or until the springtails exhibit adverse effects from the microplastic exposure. To explore the effects of microplastics on millipedes, we have begun to establish a colony of garden millipedes (Oxidus gracilis). Once established, we will use these to test microplastic exposure in a manner similar to the springtail experiments. Objective 2. Determine the effects of BDM and PE microplastic loading levels on soil community structure and function. The greenhouse experiment will be finalized, and samples collected from the experiment will be analyzed. Specifically, nematode community structure data will be analyzed, soil inorganic N (ammonium, nitrate) will be measured, and DNA will be extracted, and amplicon libraries sequenced to determine changes in community structure as a result of microplastic exposure. Soil C, microbial biomass C and enzyme analysis will be completed for all destructive sampling timepoints. Objective 3. Determine the effects of BDM and PE microplastics on microbial C cycling. The incubation experiment will be completed. In addition to completing the soil analyses (SOC, mineral N, enzymes, microbial biomass C), soil will be fractionated to particulate and mineral-associated fractions to examine the distribution of plastic-derived C in different soil pools. Final data organization and analysis will be started Soil samples from the final timepoint will be frozen at -80°C for microbial community analyses via amplicon sequencing and untargeted metabolomics to determine how these plastic feedstocks may have effect microbial communities.

Impacts
What was accomplished under these goals? Our project seeks to determine the impacts of agriculturally-relevant microplastics on soil biota and their functions. While there has been much research on microplastics, most of it involves model microplastics (e.g. spheres), and we now know that size and morphology of microplastics are key determinants in their effects on biology. To that end we are generating irregular fragmented plastics derived from agricultural plastic mulch films to determine how these more realistic microplastics affect microbial and microfaunal communities and their functions at the individual, population, and community scale. We are testing both conventional (PE) and biodegradable plastics to determine if these impacts differ between types of films. To address our goals, we are conducting experiments to address three specific objectives: Objective 1: Determine the concentration-dependent effects of BDM and PE microplastics and nanoplastics on individual soil organisms and populations. Agriculturally-relevant microplastics were produced from both conventional (PE) and biodegradable (PBAT) films using a combination of grinding and sieving to achieved a desired size range. We produced approximately 2 kg of microplastics with a size of <250 µm: particle size analysis revealed that 90% of the particles were between 100 and 250 µm. These were used across several ongoing experiments. To determine effects of microplastics on important soil microbes, we have established a small collection of bacterial and fungal strains representing key soil microbes: we had one strain from previous work (Bacillus pumulis B12), we purchased 2 strains from culture collections (Streptomyces fradiae, Rhizobium leguminosarum), and isolated 13 additional strains from soil which are identified via rRNA gene sequencing. Five of these strains (Paraburkholderia, Variovorax, Pseudomonas chlororaphis, a novel species of Agrobacterium and a Trichoderma sp. fungi) which will be used in future tests with the produced microplastics. To determine the effects of microplastics on soil nematodes, we have established a test system with root knot nematode, Meloidogyne incognita. Cucumber (Cucumis sativus) seeds were sprouted and transplanted to 13-cm conetainers to maximize nematode interaction with plant roots. The experimental treatments were 0%, 0.2%, 1.0%, and 2.0% w/w microplastics mixed into an artificial substrate of fine sand, coarse sand, and Promix in a 2:1:1 ratio. Our randomized block design include 6 replicates per treatment. After seedlings establish, we introduced 500 nematode eggs into each tube. At the conclusion of the experiment, we will harvest and stain the root systems to count nematodes that have successfully colonized the roots. Objective 2. Determine the effects of BDM and PE microplastic loading levels on soil community structure and function. To determine the effects of microplastics on communities of organisms, we began a 6-month long greenhouse study with field soils and natural communities. We mixed in either PE and PBAT microplastics at 0.2%, 1% and 2% w/w concentrations. Control pots had no microplastics added. Pots were kept under relatively constant temperature and humidity in a greenhouse. Pots were destructively samples at 1, 2, 5, 8, and 16 weeks. Soil physicochemical parameters were measured, nematode communities were extracted and counted, and aliquots of soil were flash frozen and stored at -80°C for future DNA- and metabolomic analyses. Early results show elevated respiration, decreased soil organic carbon, and elevated peptidase activity with the PBAT treatments indicating it was biodegrading. No strong changes in the nematode community richness or functional composition have been observed, but data collection and analysis is still underway. Objective 3. Determine the effects of BDM and PE microplastics on microbial C cycling. To determine effects of microplastics on soil carbon dynamics, a soil incubation experiment was begun using PE and 3 biodegradable plastic feedstocks: polybutyl succinate (PBS), polylactic acid (PLA), and a polylactic acid/polyhydroxyalkanoate (PLA/PHA) blend. These were selected because their 13C signatures were distinct from each other and also distinct from background soil, allowing us to trace plastic-derived carbon to soil carbon pools. 1 g of microplastics (<500 um) were added to 50 g soil (2% w/w). We also had a treatment with no plastic added as a control. Because we have seen from previous work that nitrogen is a major driver for C cycling, we tested nitrogen addition as a second factor with treatments being nitrogen as ammonium nitrate added at a rate equivalent of 125 kg/ha. Total and 13C-CO2 was measured at regular intervals throughout the experiment, and destructive samples were taken at 5, 10, 30, and 180 days to be analyzed for 13C, total C and N, microbial biomass carbon, mineral N, and pH. Experiment is ongoing, but early results show that PLA/PHA was decomposing at greater extent in soil compared to other BDMs, and resulting in priming of native SOC and immobilization of soil mineral N. The addition of N slowed the decomposition and priming effect.

Publications

Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Astner FA, A Gillmore, Y Yu, M Flury, JM DeBruyn, SM Schaeffer, DG Hayes. 2023. Formation, behavior, properties and impact of micro- and nanoplastics on agricultural ecosystems (a review). NanoImpact 31:100474 doi: 10.1016/j.impact.2023.100474