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