ENGINEERING PLASMA ARRAYS TO REMOVE BIOFILMS FROM FOOD PROCESSING SURFACES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1021761
• Grant No.: 2020-67018-30789
• Cumulative Award Amt.: $478,500.00
• Proposal No.: 2019-06949
• Project Start Date: Jun 1, 2020
• Project End Date: May 31, 2024
• Grant Year: 2020
• Program Code: [A1332]- Food Safety and Defense

Recipient Organization
BOISE STATE UNIVERSITY
1910 UNIVERSITY DRIVE
BOISE,ID 83725

Performing Department
ELECTRICAL & COMPUTER ENGINEER

Non Technical Summary
Foodborne pathogens create biofilms on surfaces encountered in food processing facilities. These pathogens are a significant source of contamination that threaten the food safety of the nation and result in millions of foodborne illness every year. Reducing the incidence of foodborne pathogens in food processing equipment requires harsh chemicals and factory down time. A technology that could accomplish "in-line" decontamination would save millions of dollars, decrease the incidence of foodborne illness, and reduce the use of precious water resources. Cold Atmospheric-pressure Plasmas (CAPs) use ionized gases to eradicate microbial pathogens on agriculturally important surfaces. We propose to leverage the results of our USDA seed grant to develop CAP devices that can be deployed in a variety of food industries. This project will use an engineering approach to develop several device configurations to deliver sanitizing plasma to solve problems encountered with biofilm accumulation in industry. The project has the following goals: (1) Determine the optimal conditions and mechanisms of CAP mediated antimicrobial action, (2) Construct a planar array CAP device for in line treatment of flat surfaces, (3) Design and implement a radial array CAP device. Our prior work has shown that a linear discharge CAP device can successfully kill bacterial biofilms (E. coli, Salmonella, Listeria, Pseudomonas, Staph. aureus) grown on a variety of relevant surfaces (steel, glass, plastic, rubber). Those experiments are the basis for developing CAP arrays that could be deployed in a variety of manufacturing environments to reduce chemical cleaning requirements, thus providing benefit to the food industry.

Animal Health Component

40%

Research Effort Categories

Basic

40%

Applied

40%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4010	1100	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

food safety

bacteria

biofilms

food processing

Goals / Objectives
In this standard strengthening grant application, we propose to leverage the positive results of our current USDA seed grant to develop CAP devices that could be deployed in a variety of food industries. This project will use an engineering approach to develop several device configurations to deliver sanitizing plasmas that solve common problems encountered with biofilm accumulation in industrial settings. The project has the following specific aims:Aim 1: Determine the optimal conditions and mechanisms of CAP mediated antimicrobial action. These studies will examine the role of high energy particles and reactive ion species on microbial killing and biofilm ablation (sputtering) to determine the conditions that are most efficacious for biofilm removal and decontamination on an array of surface types.Aim 2: Construct a block planar array plasma device for in line treatment of flat surfaces. In these experiments a planar array of plasma discharges will be engineered, and their anti-biofilm activity demonstrated on conveyor belt mock-ups and other industrial equipment.Aim 3: Design and implement a radial array plasma device. The device design will be optimized to deliver plasma discharges to curved surfaces such as the interior of a pipe and to demonstrate the effect of electrically biasing the target substrate on improving biofilm removal.Our seed grant sponsored work has shown that a prototype CAP device containing a single small linear plasma discharge can successfully etch away bacterial biofilms and exert antimicrobial activities against a variety of known foodborne pathogens (E. coli, Salmonella, Listeria, Pseudomonas, Staph. aureus) in biofilms grown on a variety of industrially relevant surfaces (steel, glass, plastic, rubber). Our results have shown that for all of the microbial pathogens we have examined, that we could kill >90% of microbes found in biofilms on these surfaces with a CAP exposure of less than 10 seconds. These proof-of-concept experiments lay the ground work for developing CAP arrays that can be deployed in a variety of manufacturing environments to reduce chemical cleaning requirements, thus providing a major benefit to the food industry.It is expected that this project will produce two prototype devices: one is comprised of stacked, planar plasma discharge elements to form an array over a wide area (2 x 2 cm) to treat flat food processing surfaces, and a second configuration that will deliver a radial array of plasma that could be deployed inside of fluid delivery pipes. The project will provide a training ground for numerous graduate and undergraduate students as part of an ongoing Vertically Integrated Projects interdisciplinary program. The project also strengthens the capacity of Boise State University to perform food safety research, an emerging area of study for this institution.

Project Methods
We will examine plasma device parameter effects on biofilm removal and antimicrobial activity, and investigate potential mechanisms of action.The results of these studies will allow us to visualize the effects of CAP treatment on biofilm removal, bacterial viability, and potential mechanisms by which the plasmas are exerting their effects. In the beginning, the work will utilize methods and plasma devices constructed as part of the USDA strengthening seed project, and described in the Preliminary Results section. Many of the experimental techniques described to meet Aim 1 goals will be used in Aims 2 & 3 to examine the effects of later linear-array and radial-array iterations.Biofilm preparation. Plasma design parameter effects will be studied on biofilms prepared on coupons of glass, steel, plastic, and rubber. Briefly, cultures of E. coli O157:H7 (ATCC 43894), Staphylococcus aureus (ATCC BAA-44), Pseudomonas aeruginosa (PAO-1), Salmonella enterica (var. Typhimurium), or Listeria monocytogenes (10403) are prepared by inoculating small volumes of Luria-Bertani broth (LB) or other nutrient broth from isolated bacterial colonies grown on LB agar plates. Cultures are grown overnight at 37°C with shaking at 225 rpm. To prepare biofilms on the various substrates, sterile 2.5x2.5cm coupons are inserted vertically into sterile 12-well Corning tissue culture plates, partially filled with LB broth inoculated with 10 µL overnight culture. Cultures are covered and incubated for 48 hr at 37°C with shaking at 100 rpm. Other coupon sizes, biofilm ages, and substrates can be similarly prepared as required by the experiment. Sufficient replicates will be prepared to perform all treatments in at least 3-6 times for every exposure timepoint or condition. Following incubation, the coupons are rinsed briefly in PBS to remove loosely bound planktonic cells and stored on filter papers moistened with PBS in sterile petri dishes until the plasma treatment commences (<1hr).Profilometry. To measure the ability of plasma treatment to etch (or sputter) a channel into biofilms, coupons containing 48 hr biofilms are placed 1-5 mm below the fixed position plasma discharge oriented 90° to the edge of the biofilm and treated according to the design parameters in Table 2. The etched biofilm will be examined using both a Bruker Dektax XT-A stylus profilometer and Wyko NT1100 optical profilometer available in the Idaho Microfabrication Lab (Boise State Center for Materials Characterization). Depth and width measurements of the etched channel in the biofilm will be measured to determine etch rates.Staining of etched biofilms - crystal violet (CV) staining. For larger scale removal of biofilms from surfaces produced by treatment with plasma arrays, total differences in biofilm quantity will be examined using CV staining. Briefly, following treatment the biofilm sample will be immersed in 0.1% CV solution for 15-30 min. The sample will then be rinsed gently in distilled water and airdried. CV binding to the biofilm sample will then be extracted into 30% acetic acid in water, and the absorbance of the solution at 550nm used as a measure of biofilm quantity. Final reported values will be the mean (±SEM) determined from at least 3-6 measurements of independent samples. Fluorescence staining. For some experiments, fluorescence staining will be used to prepare treated biofilm samples for microscopy to differentiate living bacterial cells (green) from dead cells (red). In these experiments, plasma treated biofilm coupons will be stained using a BacLight Live-Dead stain according to the manufacturers specifications (Molecular Probes, Eugene, OR). For total fluorescent biofilm staining, replicate samples will be stained with 0.1% acridine orange for 1hr at room temperature, and rinsed extensively prior to confocal microscopy.Microscopy of etched biofilms. Microscopy of fluorescently stained biofilm samples will be performed using a fluorescent confocal microscope available to investigators at the BSU Biomolecular Research Center. Briefly, samples will be imaged on a Zeiss LSM Meta510 microscope. At least 20 Z-stack images will be collected to assemble a 3D image of the etched/plasma treated biofilm. Additional replicate samples will be subjected to SEM analysis (BSU Microfabrication Core facility) to compare the biofilm removal pattern to results obtained by profilometry and fluorescence microscopy.CFU reduction assays. Etched biofilm coupon samples will be examined for reductions in colony forming units (CFU) as an added measure of the effect of plasma discharge on cell viability. Briefly, following treatment, biofilm coupons will be immersed in sterile phosphate buffered saline and the remaining viable cells dislodged by vortexing for 3 min with 6mm glass beads [Lindsay, 1997], and enumerated by plating onto Mueller Hinton agar plates and standard colony counts after overnight incubation at 37°C. The exposure doses (ED) required to achieve a 50% (ED50), 90% (ED90), and 99.9% reduction (3-log reduction) in bacterial viability per unit area of biofilm exposed will be calculated from dose-response curves of the data assembled from at least three independent experiments.Metabolic reduction assays. As an adjunctive to CFU reduction assays, the metabolic activity of plasma treated/etched samples will be examined using a redox sensitive resazurin dye. Briefly, plasma-treated coupons will be placed in 24 well plates containing sufficient broth media (+ 0.1% resazurin) to submerse the biofilm, and the temporal conversion dye to fluorescent resarufin (ex 560nm/em 590nm) measured using a BioTek plate reader set on top read mode as an indication of viable, metabolically active cells. The goal will be to observe a 103 (3-log) reduction in metabolic activity following plasma treatment. Data will be collected on at least 3-6 replicate samples for every treatment condition.Detection of UV. UV photons have antimicrobial activity due to their absorption and modification of a variety of cellular constituents, particularly DNA. An indication of the level of UV photons being generated by the various CAP device (linear discharges, linear array discharges, radial array discharges) will be measured using a PUV3402 ultraviolet photometer available to the co-PI (Cornell) in the department of Chemistry and Biochemistry. Measurements will be conducted in a darkroom to eliminate extraneous light and calculations of estimates of UV exposure on plasma treated samples made from these measurements. To determine how these UV exposures contribute to cell viability of plasma treated samples, a replicate series of experiments will treat biofilm coupon samples to similar wavelengths and UV doses derived from a germicidal UV lamp source (Thomas Scientific).Detection of reactive oxygen species. Reactive oxygen species (ROS) are generated in Ar/O2 plasmas, and are also a consequence of plasma interactions with air. Examples of ROS include superoxide (O2−), hydrogen peroxide (H2O2), and hydroxyl free radicals (•OH), which are all known to have antimicrobial activity. To begin to estimate the plasma production of ROS by the CAP devices, several assays will be performed. In initial experiments, 1mL samples of PBS in a 47 mm petri dish will be exposed 1 mm below the linear plasma device (or later to the plasma arrays) for increasing time spans. ROS produced by the plasma and delivered to the liquid surface will absorb and accumulate in the fluid. Periodic samples will be removed and mixed with dihydroxyphenoxazine (Amplex Red® dye, ThermoFisher), and the fluorescence that is stimulated by interaction with peroxide measured on a spectrofluorimeter (ex 57 nm/ em 585nm). Specificity will be determined by treatment of replicate samples with peroxidase (to degrade peroxide), and incorporation of known concentrations of hydrogen peroxide to establish a standard curve.

Progress 06/01/20 to 05/31/24

Outputs
Target Audience:Our teams continue to include undergraduate students working in the two laboratories: engineering and biochemistry. In addition to students directly suppoprted by this grant, other students particpated in our VIP Course:Plasma Agriculture. This last year we had 15students from biology, chemistry, and engineering in this course.We meet weekly with students to discuss their research related to bacterial biofilms. Students give presentations on journal articles they review in teh use of plasmas in sanitization. Five VIP plus laboratory student groups presented posters at ourundergraduate research conference. In addition, we are working with the new Food and Dairy Innovation Center at the university to reach our target audience. In total, more than 50 undergraduate students participated in the course over 4 years of the project. An addtional 10 undergraduate students worked in the research laboratories as undergraduate research assistants. Changes/Problems:The major problem has been the use of air as the working gas. The lower ionization cross-section of nitrogen has required operation at a higher votlage and requires more power. This high power has casued over heating of the device and test samples. Passive cooling may be required. What opportunities for training and professional development has the project provided?1. More than 50 undergraduate students have particpated in our Vertically Integrated Projects: Plasma Agriculture course over the 4 years of the project. These students are from biology, biochemistry, health sciences, electrical engineering, and mechanical engieeering. Two additonal graduate students also particpated in this course. a. Students first train in the laboratories on safety and lab practices. They are mentored by the faculty (Cornell and Browning) who run the VIP course and by graduate students. More advanced students take the course another year and also act as team leaders and mentors. b. Students are taught about bacteria and food safety issues in food processing. They review journal articles on the use of plasma for biofilm treatment and present to the class. c. Students give class oral presentations and then poster presentations in the Boise State Undergraduate Research Conference. 2. Additional udnergraduate students work in the laboratories of the PIs as part of the grant. these students work 6-8 hrs per week during the year and are trained in lab procedures and experimental efforts. The students meet weekly with the research team to go over results, troubleshoot problems, and plan for the next research projects. These students also present at the Undergraduate Research Conference. Students are mentored by team scientists and PIs. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Aim 1. Four types of plasma arrays were developed and studied: a 2.4 cm x2 cm8 element array, a radially inward 2 cm diameter 4 element array, a radially outward 2 cm diameter 4 element array,and a 10 cm x 10 cm array. Gas flow studies including measurments of flow velocity were completed to the 2.4 cm x 2 cm array and the 10 cm x 10 cm arrays. Gas manifolds, new enclosures, and shower heads were developed and implemented. This Aim was completed. Aim 2. The 2.4 cm x 2 cm array was used to inactivate several bacterial biofilms both in a fixed condition and with the samples (1 cm coupons) moving under the array. Stainless steel was primarily studied. Results showed >99% inactivation of pseudamonus fluorescens. These devices were also tested to study the reactive species. This work was concluded and showed results for quantitation of ozone (O 3 ), hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2 ), superoxide (O 2 ), hydroxyl radical (OH* ), nitric oxide (NO), and peroxynitrite (ONOO*) for various source gases such as Ar, Ar+water vapor, and air. Experiments with the 10 cm x 10 cm array using Ar have shown only modest inactivation >95% at a 1 cm gap. More experiments are need to optimize the parameters. Also, the use of air has created difficulties in over heating of the array becuase of the lower cross section for nitrogen. Aim 3. Radially outward array biofilm inactivation on PVC pipe sections havedemonstrated >99% inactivation using an Ar working gas. However, overheating has been an issue. Nevertheless, this part of the project has been completed. A radially inward array has also been demonstrated. This structure has not yet been used to studied biofilm inactivation.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: K. McCleary, D. Miller, R. Bhattacharya, M. Pearlman, K. Cornell and J. Browning, "Bacterial Biofilm Inactivation Using Cold Atmospheric Pressure Plasma," 2023 IEEE International Conference on Plasma Science (ICOPS), Santa Fe, NM, USA, 2023, pp. 1-1, doi: 10.1109/ICOPS45740.2023.10481152.
• Type: Journal Articles Status: Other Year Published: 2024 Citation: Sumona Islam, Kyle McCleary, Jose Escobosa, Daniel Miller, Dalton Miller, Jocelyn Stephens, Ranajoy Bhattachary, Don Plumlee, Ken Cornell, and Jim Browning, "Efficient Bacterial Biofilm Inactivation using Cold Atmospheric Pressure Plasma Array Sweep," to be submitted to IEEE Transactions on Radiation and Plasma Medical Sciences
• Type: Journal Articles Status: Other Year Published: 2024 Citation: Sumona Islam, Kyle McCleary, Jose Escobosa, Daniel Miller, Marcus Pearlman, Ranajoy Bhattacharya, Don Plumlee, Ken Cornell, and Jim Browning,"Improved Biofilm Etching Efficiency Using a Cold Atmospheric Pressure Plasma Device in the Food Industry," to be submitted Trends in Food Science & Technology

Progress 06/01/22 to 05/31/23

Outputs
Target Audience:Our efforts continue with our students in our VIP Course:Plasma Agriculture. This last year we had 12students from biology, chemistry, and engineering. The goal is to train students in the discipline and encourgage efforts is food safety. We meet weekly with students disucssing their research and efforts in food processing related to bacterial biofilmes. Students give presentations on jounral articles in the area.Four student groups presented posters at out undergraduate research conference, and we gave an oral presentation atthe IEEE Conference on Plasma Science. In addition, we are working with the new Food and Dairy Innovation Center at the university to reach our target audience. Changes/Problems:The etching of biofilms has been inconsistent. This has led us to redesign our gas flow structure and develop a new power supply that can provided higher plasma density at lower voltage by tuning at a resonance of the arrays. This work is ongoing. The radial arrays have also not been consistent. New designs are under developement to improve gas flow uniformity and to provide better ballast resistor configurations. The new power supply design may also help with this issue. What opportunities for training and professional development has the project provided?The project has trained 6 undergraduate students directly in engienering, biology, and biochemistry as well as 1 graduate student. In addition, another 12 undergraduate students participate in our VIP course and are training in biolfilm preparation, lab procedures, and plasma soruce operations. How have the results been disseminated to communities of interest?Presentations at the IEEE Conference on Plasma Science and at the university Undergraduate Research Conference. What do you plan to do during the next reporting period to accomplish the goals?Aim 1. Complete the etch experiments with a more consistent gas flow configuration for elements Aim 2. Demonstrate the planar array operation with a new gas flow enclosure to greatly improve flow uniformity. Align the approaches of the biology and engieeering tools to get consisten treatment. Implement a new power supply for a high power (plasma density) capability to achieve consistent etch. Aim 3. Uting the results from our planar array, create a more uniform gas flow structure and more uniform radial plasma array increased for 4 to 8 elements. Then perform new experiments in pipes to demonstrate inactivation.

Impacts
What was accomplished under these goals? Aim 1. We continued developing plasma array etch capabilties with our plasma elements. While inactivation of biofilmhas been consistent, etching of the biofilm has not. Experiments with different gas flows and oeprating conditions sometimes show clear removal, but this varies. We have also determined that our gas flow rate is not uniform. We are restructuring this in our arrays. Aim 2. We continue to improve our arrays. We currently used discrete ballast resistors for the planar arrays providing greater uniformity. A new gas flow enclosure has been developed and is now being implemented to improve gas flow uniformity. Experiments on an XY stage do not show stong inactivation. This problems is believed relevant to biofilm drying as similar experiments in our companion biology setup show very consistent inactivationresults. These experiments are being replicated in parralel (engineering and biology setups) to find the issue. Measurements of the reactive oxygen and nitrogen species with the array are complete. These experiments clearly show which species are generated and in what concentrations relative to the source gases (Ar, N2, air, O2, water vapor). These efforts have been a m,ajor success of the program. Finally, we have developed larger array elements (10 cm long) which are being used for even greater coverage. This work will continue. Aim 3. The radial arrays have been studied in simple cylindrical encolusres (PVC pipe sections). Preliminary results are enourgaging, but the resuslts are on ~70-90% inactivation compared to planar arrays which can achieve >99% inactivation. The experimental setup needs improvement for better alignment and sample handling.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: M Pearlman, M Okebiorun, C Waite, D Miller, T Koch, J Escoba, J Tenorio, D Plumlee, K Cornell, J Browning, "Biofilm Remediation Using Cold Atmospheric Pressure Plasma Planar and Radial Arrays," IEEE Conference on Plasma Science (2022). DOI: 10.1109/ICOPS45751.2022.9813128

Progress 06/01/21 to 05/31/22

Outputs
Target Audience:Our efforts primarily focused on students (undergraduate and graduate) in our VIP Course:Plasma Agriculture. This last year we had 15 students from biology, chemistry, and engienering. The goal is to train students in the discipline and encourgage efforts is food safety. We met weekly with students. Weprovided a poster presentation at the IEEE Conference on Plasma Science. We also gave a radio interview on the local camus radio station regarding our efforts. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The porjects have provided numerous training for electrical and mechanical engineering students to design and build devices, operate devices, run experiments, program data acquisition, and analyze data. 7 different undergraduate students particpated in the project. Biology and biochemistry students were trained to generate and deposit bacterial biofilms on surfaces including all safety and lab protocols. Students then operated the planar and radial arrsy to look at killing and the study of plasma chemistry through several techniques. The ran experiments and analyzed data. 5 undergraduate students and 1 graduate student worked on the project. All students learned to present data and give presentations. The graduate student won the western regional "3 minute" thesis award. How have the results been disseminated to communities of interest?In addtion to conferences and student presentations, we have given a radio interview on the research. What do you plan to do during the next reporting period to accomplish the goals?1. Determine the best plasma operation conditions to get the fastest etch at a 5 mm plasma to traget distance using argon and air. 2. Implement next generation planar and radial arrsy using these results to demonstrate rapid biofilm removal over large surface areas using an XY stage.

Impacts
What was accomplished under these goals? 1. We have begun optimizing etch conditions based on plasma oeprating parameters (voltage, current, gas flow and mixture, source geometry). We have foundthat water vapor in the Argon is important to etching. We have also determined that the resistance used on the low votlage side of the device is critical in generating an intense plasma, but plasma uniformity then becomes an issue. This configuration must be studied and used in planar arrays. 2. WE have made major fabrication changes to our planar array. The discharge gap spacer material has been replaced to proviude easier assembly and simpler fabrication. The integrated ballast resistor was a major issue. Uniformity of resistance could not be consistently achieved. The device was redesigned to allow for component, flat resistors. Using this methode, we have created more uniform and stable arrays. Determining the plasma chemistry (species) that affects killing and etch have been a major part of the research. We have now completed testing of 2.4x2 cm arrays to measure the reactive oxygen species based on the gas mixtures (Ar, Ar/O2, Air, Air/water vapor, Ar+Water vapor). This work will not extend to the best etching parameters. 3. We have completed 2nd generation radial array structures. Orginal devices suffered form overheating. The new designs still do not have ballast resistors, so uniformity is an issue. Preliniary experiments using radial arrays to kill biolfim on "pipe" surfaces has been completed. Initial results are not so good as with flat surfaces. Experimental setup needs improvement (better structure alignment).

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Adam Croteau, Amanda White, Kenneth A. Cornell, and Jim Browning, "Cold Atmospheric Pressure Plasma Device Exhibits Etching Effects on Bacterial Biofilms," s IEEE Trans. on Nuclear and Plasma Medicine, Vol 6 (2022) DOI: 10.1109/TRPMS.2021.3133183
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Adam Croteau, Spencer Goering, Dalton Miller, Sam Clark, Amanda White, Jose Escobosa, Ryan Olson, Cade Solich, Serena Stranger, Brenden Bjorklund, Kalynn Alexander, Marcus Pearlman, Ken Cornell, Don Plumlee, Jim Browning, "Cold Atmopsheric Pressure Plasma Planar and Radial Arrays for Biofilm Remediation," IEEE Conference on Plasma Science (2021). https://doi.org/10.1109/ICOPS36761.2021.9588486

Progress 06/01/20 to 05/31/21

Outputs
Target Audience:Our efforts primarily focused on students (undergraduate and graduate) in our VIP Course:Plasma Agriculture. The goal is to train students in the discipline and encourgage efforts is food safety. We met weekly with students. A seminar was presented on the research in the Electrical and Computer Engineering department tp reach students in engineering. An invited talk was given at the IEEE Conference on Plasma Science on the research and application in Food safety. Changes/Problems:1. The gap beween the plasma source and substrate is too small (< 2 mm) for best results. This is an issue for practical application. Improving performance may require redesigning the electrode geometry or oeprating at higher discharge gaps wol allow higher power operation. What opportunities for training and professional development has the project provided?We have used educational and other training activities. Research Mentoring-- multiple udnergraduate students in biochemistry, biology, electrical engineering, and mechanical engineering have performed research under this effort. These students were mentored by the faculty researchers and graduate students. They worked directly on the projects building plasma devices, oerpating the devices, preparing biological samples, and analyzing results. They were required to present at an undergraduate research conference. VIP Course-- our VIP course with 12 students met regularly over the year (fall and spring) with training in plasma science, biological sample preparation, operatiionof the plasma experiments, and research activites. They kept electronic notebooks, gave oral presentations, presented at an undergradaute research conference, and analyzed published journal articles. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Aim 1. We will refine our reactive species diagnostics and then begin careful correlation of plasma array etch rate with reactive species generation. Aim 2. We will improve the uniformity and performance of the array by using better ballasting (resistors) and by further developing our high intensity discharges. The goal this year is to demonstrate biofilm etch (5 um) on a steel substrate in a 2.4x 2 cm area within 30 s. This requires understanding the reactive species (Aim 1) as well as power coupling tothe plasma. Finally, the discharge works best with a gap of < 2 mm. This needs to be increased to 5 mm with hohger power coupling. Aim 3. The radial array elements will be redesigned with internal ballast resistors and set in segmented sections to minimize RF coupling. Then these arrays (5 element) will be used to kill and then remove biofilm from inside well plates using a removable steel coupon.

Impacts
What was accomplished under these goals? Aim 1: We have begun studies of teh different reactive oxygen species that are necessary to kill/remove biofils. We have measured OH radicals, peroxide, and ozone levels vs plasma exposure time and intensity. A clear correlation with peroxide generation is developing, but more array work is needed. WE have show biofilm removal (etch) of psuedamonas fluoresence using a linear dicharge with water vapor, indicating water generated peroxide is key. Enhancing etch rate is ongoing Aim 2: We have fabricated an 8 element plasma array (2.4x2cm) and have demonstrated the ability to kill bacterial biofilms and planktonic cells on steel substrates.However, we have not yet demosntrated the same level of etching as with a single line discharge. A new power supply will help with this activity. We have also been developing a more intense discharge source, which we plan to use for biofilm removal after further development Aim 3: We have built and demonstrated a 4 cm diameter plasma radial array of 3 elements. Uniformity of discharge and power consumption (too high) still must be optimized through ballast resistance and design changes. However, prelimionary results are very encouraging.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: 1. KA Cornell, A White, A Croteau, J Carlson, Z Kennedy, D Miller, M Provost, Spencer Goering, Don Plumlee, Jim Browning, Fabrication and Performance of a Multidischarge Cold-Atmospheric Pressure Plasma Array, IEEE Tran. Plasma Sci., Vol. 49, pp. 1388-1395 (2021) https://doi.org/10.1109/TPS.2021.3064993
• Type: Journal Articles Status: Published Year Published: 2020 Citation: 7. Kenneth A Cornell, Kate Benfield, Tiffany Berntsen, Jenna Clingerman, Adam Croteau, Spencer Goering, Daniel Moyer, Mariah Provost, Amanda White, Don Plumlee, Julia T Oxford, Jim Browning, A Cold Atmospheric Pressure Plasma Discharge Device Exerts Antimicrobial Effects, International Journal of Latest Trends in Engineering & Technology: IJLTET, Vol. 15, PP. 36-41 (2020).
• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Adam Croteau, Amanda White, Kenneth A. Cornell, and Jim Browning, "Cold Atmospheric Pressure Plasma Device Exhibits Etching Effects on Bacterial Biofilms," submitted to IEEE Trans. on Nuclear and Plasma Medicine (April 2021)
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: INVITED: 1. Jim Browning, Adam Croteau, Amanda White, Zeke Kennedy, Jessica Carlson, Spencer Goering, Mariah Provost, Madison Sullivan, Ken Cornell, Don Plumlee, COLD ATMOSPHERIC PRESSURE PLASMA ARRAY FOR BIOFILM INACTIVATION, 47th International Conference on Plasma Science 2020, 24-28 May 2020 (postponed to December 2020), Marina Bay Sands, Singapore.