Progress 07/01/16 to 06/30/21
Outputs
Target Audience:Target Audience: Interdisciplinary scientific community spanning engineering, agriculture, biology, soils and plant physiology. Changes/Problems:Despite the challenges of the Covid-19 Pandemic the PI and graduate students were able to continue research and have access to shared core facilities on the USU campus. Some delays were encountered through required isolation and quarantining. What opportunities for training and professional development has the project provided?Professional development opportunities have been provided through multiple interdisciplinary interactions during the course of this 5-year project. Several visiting scholars in the PI and collaborator labs have provided training and insight to the students working on this project. These included two graduate student visiting scholars from Brazil and Taiwan, and a sabbatical stay from a Professor from South Korea. The PI lab hosts summer high school and undergraduate students who learn research from the PI, graduate students, and undergraduate students in the lab. Many of these outreach students choose summer projects overlapping with the research goals of understanding bacteria biofilms benefiting plants. How have the results been disseminated to communities of interest?Over the 5-year period results have been disseminated in peer-reviewed journal articles, conferences presentation and proceedings, and student theses (MS completed, PhD nearing completion). What do you plan to do during the next reporting period to accomplish the goals?Nothing to report
Impacts
What was accomplished under these goals?
The major goals of this 5-year project were addressed through a focus on the plant pro-biotic root-colonizing organism, PcO6, isolated from dryland wheat in Cache Valley, Utah. The engineering design of an artificial root system for growing PcO6 biofilms and isolating OMVs was developed and validated. OMV yields were sufficient for physiochemical characterization using SEM, AFM, DLS, zeta potential and Raman spectroscopy. Yields were also sufficient for OMV / membrane interactions conducted in micro-wells of a Langmuir film balance using tensiometry to report adsorption-induced changes in the surface pressure of lipid monolayers at the air/water interface. Purified OMV yields were insufficient for an assessment of bioactivity in planted wheat. Additional methods, including tangential flow filtration, size exclusion chromatography, and field flow fractionation have been reviewed as methods for increasing isolation yields as part of review paper resulting from this research. These accomplishments are detailed and disseminated in a variety of outputs, including peer-reviewed journal articles, conferences presentation and proceedings, and student theses (MS completed, PhD nearing completion).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
"Surface characterization of outer membrane vesicles, naturally-produced colloids, from bacterial biofilms" Matthew Potter, Anne Anderson, David Britt. 95th ACS Colloid and Surface Science Symposium (Virtual) 14-16 June 2021
https://sites.psu.edu/2021colloids/
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
"Copper oxide nanoparticles and the outer membrane vesicles of a plant health promoting rhizobacterium" Matthew Potter, Cynthia Hanson, Anne Anderson, Elizabeth Vargis, David W Britt. Sustainable Nanotechnology Organization (SNO) 9th Annual Conference. November 12, 2020 - November 13, 2020 (Virtual)
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Britt, D.W. (2021, July 08). Plug-and-play bioinspired seed coatings. Nature Food, 2(7), 456-457.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Potter, M., Deakin, J., Cartwright, A., Hortin, J., Sparks, D., Anderson, A. J., McLean, J. E., Jacobson, A., & Britt, D.W. (2021, May 19). Absence of Nanoparticle-Induced Drought Tolerance in Nutrient Sufficient Wheat Seedlings. Environmental Science and Technology.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2021
Citation:
Presentations
Potter, M. K. (Presenter & Author), Anderson, A. J. (Author Only), Britt, D. W. (Author Only), 95th ACS Colloid and Surface Symposium, "Surface characterization of outer membrane vesicles, biologically-produced colloids, from bacterial biofilms," American Chemical Society, Virtual. (June 14, 2021 - June 16, 2021)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
Presentations
Potter, M. (Presenter & Author), Hansen, C. (Author Only), Vargis, E. (Author Only), Jacobson, A. (Author Only), McLean, J. E. (Author Only), Anderson, A. J. (Author Only), Britt, D. W. (Author Only), Sustainable Nanotechnology Organization (SNO) 9th Annual Conference, "Copper oxide nanoparticles and the outer membrane vesicles of a plant health promoting rhizobacterium." (November 12, 2020 - November 13, 2020)
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Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Academic, through professional society presentations and publications. Changes/Problems:Some minor disruptions of activity were associated with the COVID-19 Pandemic. The primary Ph.D. student working on the project had non-COVID related health issues contributing to some delays. What opportunities for training and professional development has the project provided?Two Ph.D. students have been working together to improve the design of the root mimetic system. A summer outreach student from the USU Blanding Campus was trained by the graduate students during her 10-week research engagement in the lab. She learned basic microbiology, microbe-plant interactions, particle / vesicle size characterization techniques, and assessment of plant health using a portable photosynthesis unit. How have the results been disseminated to communities of interest?The OMV work has been published in a student's M.S. thesis which is now publicly available on Utah State's digital commons (https://digitalcommons.usu.edu/etd/7864/). The OMV work, with the additional LDA experiments, was prepared as a manuscript and published in "Scientific Reports", an open-access journal (https://rdcu.be/cbHPV). The research was presented to the general scientific community in the 9th Annual Sustainable Nanotechnology Organization (SNO) Conference. What do you plan to do during the next reporting period to accomplish the goals?The lumen-fed root mimetic system will be improved to study PcO6 growth as a function of droughted or watered wheat artificial root exudates. Our current design for the system has struggled with consistent delivery of the nutrients through the lumen. Details such as tube diameter, fluid pathway, and syringe pump calibration will be adjusted to ensure consistency and repeatability in the root mimetic experiments.Chemical characteristics of PcO6 biofilms grown on a lumen-fed root mimetic system will be measured using biochemical assays including assays measuring DNA, protein, and polysaccharide concentrations. These concentrations will be normalized to the colony forming units (CFUs) within the biofilm.Chemotaxis of PcO6 towards the artificial root exudates will be explored using three fluorescent PcO6 constructs- red fluorescent protein (RFP), green fluorescent protein (GFP), and cyan fluorescent protein (CFP). The three strains will be inoculated on a root mimetic with one in the fluid matrix, one on the distal end of the root, and one on the proximal end of the root.The communication potential of OMVs will be tested by exposing biofilm deficient PcO6 mutants to OMVs of wild type PcO6. Changes in the production of proteins with a bicinchoninic acid assay, DNA with a PicoGreen dsDNA quantification assay, polysaccharides with a method from the literature [1], and other components of biofilm matrices will be measured.OMVs will be further characterized with these same assays. OMV production will be quantified using the lipophilic dye FM4-64. While Raman spectroscopy is a concentration-independent technique, other characterization techniques measuring proteins, DNA, polysaccharides, and other biomolecules will require a baseline for normalization.
Impacts
What was accomplished under these goals?
Linear discriminant analysis (LDA) is one method to quantitatively view differences in Raman spectra. This machine learning technique creates an algorithm to sort Raman spectra according to the treatment then tests the accuracy of the algorithm by predicting the treatment of each spectrum. The actual and predicted treatments are compared. This was previously done on Raman spectra of Pseudomonas chlororaphisO6 (PcO6) biofilm cells and isolated OMVs with accuracies of 83.3% and 77.1% respectively. To determine what portions of the spectra were used for these groupings, LDA was performed on smaller portions of the spectra. For both the biofilm PcO6 cells and OMVs, groupings were largely dependent on the 750-1500 cm-1 region, where most Raman peaks are located. Further narrowing the spectral regions for PcO6 biofilm cells, spectra groupings appeared to be largely dependent on the 1400-1500 cm-1 and 1500-1700 cm-1 regions. The1400-1500 cm-1 region contains several protein signatures. The 1500-1700 cm-1 region contains various amino acid, nucleic acid, amide, and lipid signatures. In contrast, LDA of narrower portions did not vary greatly between the spectra portions in the 750-1500 cm-1 region (83-89% accuracy) meaning that LDA groupings were less dependent on single peaks/peak sections. These results show the importance of quantitative techniques when examining spectroscopy data. Wheat exudates were measured for wheat (Triticum aestivum cv. Juniper) under drought and watered conditions. Artificial root exudate (ARE) solutions were mixed based on these results. PcO6 was grown on a solid medium of AREs mixed with a quarter-strength Hoagland's solution. Hoagland's solution was chosen for an amendment as the artificial root exudate lacked phosphorus and nitrogen, both of which are essential for bacterial growth. PcO6 also successfully formed biofilms on a lumen-fed root mimetic system with artificial root exudates fed through the lumen of the membrane and quarter-strength Hoagland's solution in the exterior of the chamber. These first steps will lead to understanding plant-bacteria relationships and how these relationships are altered during drought.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Cartwright, A., Jackson, K., Morgan, C., Anderson, A. J., & Britt, D. W. (2020, July 01). A review of metal and metal-oxide nanoparticle coating technologies to inhibit agglomeration and increase bioactivity for agricultural applications. Agronomy, 10(7).
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Potter, M., Hanson, C., Anderson, A. J., Vargis, E., & Britt, D. W. (2020, December 01). Abiotic stressors impact outer membrane vesicle composition in a beneficial rhizobacterium: Raman spectroscopy characterization. Nature: Scientific Reports, 10(1).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
Presentations
Cartwright, A. (Presenter & Author), Eskeets, J. (Author Only), Jacobson, A. (Author Only), McLean, J. E. (Author Only), Anderson, A. J. (Author Only), Britt, D. W. (Author Only), Sustainable Nanotechnology Organization (SNO) 9th Annual Conference, "5 nm Pluronic F68 Micelles and Glycine Betaine Increase Wheat Seedling Growth and Water Content During Drought." (November 12, 2020 - November 13, 2020)
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Academic, through professional society presentations and publications. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The graduate student working on this project successfully defended his M.S. thesis on this research and has been admitted into the Ph.D. program in Biological Engineering. How have the results been disseminated to communities of interest?The research was presented as an M.S. thesis defense open to the university community. The thesis will be archived in Digital Commons, and a manuscript will be submitted based on the OMV work. What do you plan to do during the next reporting period to accomplish the goals?Raman spectroscopy with linear discriminate analysis of OMVs will continue for OMVs isolated from biofilms receiving artificial root exudates as nutrients. This will allow for correlations between OMV properties and host plant health to be identified. The artificial root exudate compositions match those from characterized wheat root exudates. The ability of OMVs to stimulate biofilm growth in PcO6 will be explored using wild type and mutants deficient in specific aspects of biofilm formation. Modes of OMV interaction and uptake by the plant host will also be explored using fluorescent protein producing PcO6.
Impacts
What was accomplished under these goals?
OMVs were purified from biofilm cells of the plant-health-promoting rhizobacterium Pseudomonas chlororaphis O6 (PcO6). Two relevant rhizosphere stressors were examined: CuO nanoparticles (NPs), a fertilizer and fungicide, and H2O2, generated during plant stress responses. Atomic force microscopy revealed 40-300 nm diameter OMVs from control and stressed cells. Raman spectroscopy with linear discriminant analysis (LDA) identified PcO6 cells according to stressor and resultant purified OMVs with 83.3% and 77.1% accuracies, respectively. All OMVs had higher relative concentrations of proteins, lipids, and nucleic acids than intact PcO6 cells. Nucleic acid concentration increased in OMVs isolated from stressed cells: CuO NP-induced OMVs > H2O2-induced OMVs > control OMVs. Biochemical assays confirmed the presence of lipopolysaccharides, nucleic acids, and protein in OMVs; however, these assays could not discriminate OMVs according to cellular stressor. These results demonstrate the utility of Raman spectroscopy and LDA to distinguish among OMVs from control and stressed cells and provide insight into the macromolecules packaged into OMVs relating to bacterial stress responses.
Publications
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Academic, through professional society presentations and publications. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The post-doctoral associate provided the graduate student in-depth training on Raman spectroscopy. The graduate student also received training in density gradient ultracentrifugation. The graduate student presented this work at two conferences focusing on nanotechnology, allowing for interaction with peers and established experts in this field. How have the results been disseminated to communities of interest?Results were presented as a poster at a Gordon Research Seminar (June 2018), a poster at a Gordon Research Conference (June 2018), and a podium presentation at the annual meeting of the Sustainable Nanotechnology Organization (SNO, November 2018). What do you plan to do during the next reporting period to accomplish the goals?The plan of work will focus on linear discriminant analysis of Raman spectra of OMVs collected for control and the abiotic stressor of H2O2 and CuO nanoparticle. These will be compared to Raman for PcO6 cells under the same conditions to identify if selective subset of compounds induced by the stressors in the whole cells are packaged into/on the OMVs. Peak assignments in Raman will be supported by OMV biomolecule quantification, including protein, lipid, and DNA content.
Impacts
What was accomplished under these goals?
Outer membrane vesicles (OMVs) were isolated from the beneficial root-colonizing microbe, Pseudomonas chlororaphis O6 (PcO6) under baseline and external stressor conditions. Bacteria where grown as biofilms in the form of bacterial lawns on rich medium (LB) growth plates and subject to hydrogen peroxide (3%) or CuO nanoparticle (30 mg/L) challenges or double distilled water (negative control). Isolation of OMVs from bacterial biofilms is an advancement over the standard protocols using liquid growth (planktonic) conditions. The OMVs were isolated using density gradient ultracentrifugation methods and analyzed with dynamic light scattering and Raman spectroscopy. DLS reported hydrodynamic diameters of OMVs between 150 and 300 nm for all OMV isolates. Raman spectroscopy showed distinct chemical signatures for the OMVs isolated under these different stressors using least discriminate analysis. The Raman for OMVs were contrasted with Raman analysis of whole PcO6 under the same stressor conditions. All OMVs fall within, or just above, the dimensions of nanoparticles (NPs). Linear discriminant analysis shows that exposure to CuO NPs or H2O2 alters the Raman spectra of PcO6 bacteria. This may indicate differences in secretions, including OMVs, from PcO6 cells under these abiotic stressors. The OMVs of PcO6 exposed to CuO NPs or H2O2 show differences in peak intensity of Raman spectra compared to OMVs of PcO6 without stressors. Both PcO6 cells and OMVs show peaks for similar compounds including nucleic acids, amino acids, proteins, lipids and carbohydrates An aromatic carbon peak (3064 cm-1) present in Raman spectra of PcO6 cells is noticeably absent in Raman spectra of OMVs. Differences in intensity of peaks of PcO6 compared to OMVs suggests differences in relative concentrations of compounds.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Presentations
Potter, M. K. (Presenter & Author), Bahe, C. (Author Only), Jacobson, A. (Author Only), Anderson, A. J., McLean, J. E. (Author Only), Britt, D. W. (Author Only), Sustainable Nanotechnology Organization, "CuO nanoparticle influence on wheat photosynthetic efficiency during simulated drought," Washington D.C. (November 14, 2018 - November 17, 2018)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Presentations
Potter, M. K. (Presenter & Author), Hanson, C. (Author Only), Anderson, A. J. (Author Only), Vargis, E. (Author Only), Britt, D. W. (Author Only), Sustainable Nanotechnology Organization, "Nanoparticle influence on bacterial outer membrane vesicles," Washington D.C. (November 14, 2018 - November 17, 2018)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Presentations
Cartwright, A. (Presenter & Author), Kunzler, D. (Author Only), Morgan, C. (Author Only), McLean, J. E. (Author Only), Jacobson, A. (Author Only), Anderson, A. J., Britt, D. W. (Author Only), Sustainable Nanotechnology Organization, "Protective osmolyte coronal layers to enhance nanoparticle bioavailability and activity," Washington D.C. (November 14, 2018 - November 17, 2018)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Presentations
Cartwright, A. (Presenter & Author), McLean, J. E. (Author Only), Anderson, A. J. (Author Only), Jacobson, A. (Author Only), Giasuddin, A. B. M. (Author Only), Kunzler, D. (Author Only), Morgan, C. (Author Only), Valiente, J., Britt, D. W., Gordon Research Conference: Nanoscale science and engineering for agriculture and food, "A novel root mimetic platform for testing the effects of SiO2 nanoparticles on the architecture of beneficial biofilms," GRC, Mount Holyoke, South Hadley, MA. (June 3, 2018 - June 8, 2018)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Presentations
Potter, M. (Presenter & Author), Hanson, C. (Author Only), Vargis, E. (Author Only), Britt, D. W., Gordon Research Conference: Nanoscale science and engineering for agriculture and food, "Bacterial outer membrane vesicles: Natures nano," GRC, Mount Holyoke, South Hadley, MA. (June 3, 2018 - June 8, 2018)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Presentations
Morgan, C. (Presenter & Author), Cartwright, A. (Author Only), Kunzler, D. (Author Only), Britt, D. W., Gordon Research Seminar: Nanoscale science and engineering for agriculture and food, "Capped nanoparticles improving controlled nutrient delivery to crops," GRC / GRS, Mount Holyoke, South Hadley, MA. (June 3, 2018 - June 4, 2018)
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Academic, through professional society presentations and publications. Changes/Problems:An additional analytical method of Raman spectroscopy will be applied to characterize the isolated OMVs. What opportunities for training and professional development has the project provided?SEM training was completed for a graduate student through the USU Core Microscopy Facility. Nine students from 2-year colleges attending a summer undergraduate research experience in Biological Engineering at USU gained fundamental skills in biological engineering, microbiology, plant growth, and access to advanced analytical equipment, including SEM. How have the results been disseminated to communities of interest?One journal article was published reporting on the development of the root mimetic system and applications. Results were presented as posters at three national conferences. What do you plan to do during the next reporting period to accomplish the goals?Using the developed root mimetic system bacteria biofilms will be assessed for abiotic stress responses, with the aim of inducing OMV production. Standard methods of TFF and centrifugation will be employed for OMVs isolation. Isolation methods will be optimized to provide maximum OMV yields for subsequent characterization.
Impacts
What was accomplished under these goals?
Development of an artificial root mimetic using a porous hollow fiber membrane. Determination of biofilm growth as a function of artificial root exudate carbon and nitrogen content up to 1 week. Bacterial biofilm formation assessed using two different plant beneficial microbes. Established biofilms were examined using scanning electron microscopy to assess bacterial response to CuO and ZnO nanoparticle challenges as abiotic stressors. Spherical granules were identified within the PcO6 bacteria comprising the biofilms that had not previously been reported for this microbe. Putative outer membrane vesicles (OMVs) were also identified in the biofilms using SEM.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Potter, M. (Presenter & Author), Doxie, S. (Author Only), McLean, J. E. (Author Only), Anderson, A. J. (Author Only), Jacobson, A. (Author Only), Britt, D. W. (Author Only), Sustainable Nanotechnology Organization, "CuO Nanoparticle Modified Lignification of Wheat," Los Angeles, California. (November 5, 2017 - November 7, 2017)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Britt, D. W. (Presenter & Author), Mohammad Giasuddin, A. B. (Author Only), Sustainable Nanotechnology Organization, "Self-assembly of tri-functional and di-functional alkane silanes into hydrophobic silica nanoparticles in aqueous media," Los Angeles, California. (November 5, 2017 - November 7, 2017)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Britt, D. W. (Presenter & Author), Potter, M. (Author Only), Anderson, A. J. (Author Only), Villanueva, I. (Author Only), Taylor, T. A. (Author Only), Sustainable Nanotechnology Organization, "Summer education in nano- and biological approaches to protect plants against drought stress," Los Angeles, California. (November 5, 2017 - November 7, 2017)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Bonebrake, M. (Presenter & Author), Anderson, K. (Author Only), McLean, J. E., Jacobson, A., Anderson, A. J., Britt, D. W., Institute of Biological Engineering, "Characterization of biofilms and metabolites in a synthetic rhizosphere," IBE, Salt Lake City. (March 30, 2017 - April 1, 2017)
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Progress 07/01/16 to 09/30/16
Outputs
Target Audience:Target Audience Scientific community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Opportunities One MS student has been recruited for this project (Michelle Bonebrake) and she has received training from the project director on fundamental microfluidic delivery systems needed for the root-mimetic HFM system. She has also been trained on fluorescence and brightfield microscopy methods used for biofilm characterization. She has developed several prototype root-mimetic platforms. A second MS student (Kaitlyn Anderson) whose research focused on biomedical aspects of HFMs has provided assistance with the root-mimetic development. An undergraduate student volunteer (Matthew Potter) has provided additional assistance by developing methods to potentially create root-mimetics using root epidermal cell sheaths. All students are in the Biological Engineering Department at USU. Training in bacterial culture and plant growth methods to the students was provided by Dr. Anne Anderson in the USU Biology Department. How have the results been disseminated to communities of interest?Dissemination None yet. Dissemination of the preliminary work at the annual Institute of Biological Engineering conference, to be held in SLC, April 1-2, 2017, is planned. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work Using the root-mimetic HFM system the role of outer membrane vesicles (OMVs) as carriers for secondary metabolites will be characterized as a function of rhizoexudate / defined media delivered through the fiber lumens. Fluorescence, brightfield, and scanning electron microscopy methods will be employed to characterize biofilm architectures under baseline and stress response conditions. Methods of controlling the delivery rate of root exudate using gravity feed and capillary action will be investigated as alternatives to syringe pump delivery. Single and mixed component bacterial films of PcO6 and Bs will be investigated for biofilm forming potential and OMV production and cross-communication under baseline and stressed conditions, simulated by different rhizoexudate compositions.
Impacts
What was accomplished under these goals?
Accomplishments A root-mimetic model using a hollow fiber membrane (HFM) filter was developed and validated for two root-colonizing bacteria, Pseudomonas chlorororaphis O6 (PcO6) and Bacillus subtilis (Bs), both isolated from wheat. The root-mimetic system has demonstrated robust bacterial biofilm formation in a simulated rhizosphere. The biofilms were fed defined media representing subsets of rhizoexudates, allowing for the assessment of plant components on the biofilm architecture and production of secondary metabolites.
Publications
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