Progress 06/01/18 to 11/30/19
Outputs
Target Audience:Food safety is an incredibly important aspect of the food supply. The contamination of food and food processing equipment by food-borne pathogens such as Listeria and Salmonella (as well as many others) are a major concern resulting in lost food, illness including deaths, and significant economic loss. According to the USDA, every year approximately 48 million people become ill from foodborne pathogens resulting in costs of over $15.5B annually and thousands of deaths. Bacterial colonies called biofilms can build-up on equipment and food. These biofilms are extremely difficult to remove. Methods to kill pathogens are varied and depend upon the food type, harvesting, and processing. While some of these methods are effective, some food types (e.g. spinach) are difficult to treat, and some processes (e.g. steam cleaning) use significant amounts of water and power. In addition, some processes use harsh chemicals which can affect both the food quality and the food workers. Therefore, more effective, low cost, practical, and environmentally effective processes are desirable. One possible method uses Cold Atmospheric Pressure Plasma (CAP). A plasma is an ionized gas in which gas molecules such as oxygen and nitrogen (components of air) are stripped of their electrons resulting in positive ions. The ions of oxygen can be used to kill and physically remove bacteria on food and on food processing equipment without resulting to harsh chemicals. Plasma is used routinely in the semiconductor industry to fabricate microelectronics. More recently plasma has been used in agriculture. The area of Plasma Agriculture has grown in the last 20 years with numerous demonstrations of CAP treatment on a variety of food products. There are numerous types of sources which have been used to treat a wide variety of food products as well as pathogens responsible for human illnesses. In the agricultural field, CAP has been used to kill Listeria on meat surfaces, to kill Salmonella on fresh produce, and to inactivate biofilms and biofilm-forming bacteria. However, there are still issues to be resolved concerning CAP efficacy, effects on food quality, and integration into practical systems for cost-effective food processing. Our primary goal is to develop a new type of CAP that can be used in a variety of food processing settings for both treating food and cleaning food processing equipment. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has involved 18 total undergraduate students and 2graduate students in a variety of training opportunities. 7 total undergraduate students were supported for research on this grant. Two additional students worked on this project leveraging Ralph Jones Fellowship support during 2018/2019. Their efforts included training on the fabrication, operation, and testing of the plasma sources. An additional eight students participated in the Vertically Integrated Projects (VIP) Plasma course. This course met for 1 credit hour in the fall of 2018, 2 credit hours in the spring of 2019, and again for 1 credit in the fall of 2019.. Students in this course were trained on the testing and use of the plasma source, and were trained on the preparation of biofilms on a variety of solid substrate using cultures of foodborne pathogens. They exposed these samples to the plasma source, and then were trained to perform colony forming unit (CFU) course. In addition students: 1. Prepared laboratory notebooks 2. Were trained in giving oral presentations and then presented multiple times on their research in the course 3. Presented posters at several undergraduate research conferences 4. Wrote research reports 5. Dr. Browning presented the results of this work at the IEEE Conference on Plasma Science (June 2018 and June 2019) and graduate student Adam Croteau also present at the ICOPS (June, 2019). While at the conference both partipants attended sessions on applications of plasma sources in industrial and agricultural environments. He also delivered a seminar on the topic during the Electrical and Computer Engineering Department seminar series in spring 2019. How have the results been disseminated to communities of interest?The results of our investigations have been presented at sevenconferences to a wide audience of professional, student, and community members. These conferences include: 2018 IEEE Conference on Plasma Science (Denver, CO, June 2018) 2018 Idaho Council on Undergraduate Research (Boise, ID, July 2018) 2018 Idaho INBRE Summer Research Conference (Moscow, ID, August 2018) 2019 BSU Undergraduate Research Conference (Boise, ID, April 2018) In addition, our work was presented to members of the public during the 2019 Bronco Days during tours of the research facilities conducted by our students. 2019IEEE Conference on Plasma Science (Orlando, FL2019). 2 posters What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The team demonstrated a reduction of food borne pathogens (E. coli, Pseudomonas, Salmonella, Staphylococcus) on a variety of substrates (glass, steel, and plastic) using our Cold Atmopsheric pressure Plasma (CAP) sourc. All bacteria are reduced by 90% or greater after 30 s of plasma exposure based on Colony Forming Unit (CFU) counts. These experiments were performed using a ingle line discharge (25mm x 0.5 mm) with a rotating biofilm sample for coverage. Our team has also develop a CAP array with 8 elements. Each discharge element is connected to an internal ballast resistor to control the discharge current and improve uniformity. Experiments using external resistors from 100 kOhmto 200 kOhm show very good discharge uniformity above 100 kOhms. CAP arrays are currently being tested to demonstrated biolm deactivation on substrates. Preliminary results continue to demonstrate CFU reduction of >90%, and the CAP arrays do not require physical movement to provide 100% area coverage (24 x 24 mm).
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2019
Citation:
Adam Croteau, Joe McCarver, Zeke Kennedy, Amanda White, Ken Cornell, and Jim Browning, Statistical Quantization and Optimization of Cold Atmospheric Pressure Plasma Source
for Destroying Bacteria and Biofilms through Design of Experiments Method
, 45th IEEE International Conference on Plasma Science (ICOPS 2019), Orlando,FL USA (2019)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Kenneth A. Cornell, Kate Benfield, Tiffany Berntsen, Jenna Clingerman, Adam Croteau, Spencer Goering, Daniel Moyer, Mariah Provost, Amanda White, Julia T. Oxford, Don Plumlee, and Jim Browning, "A Cold Atmospheric Pressure Plasma Discharge Device Exerts Potent Antibiofilm Effects," submitted to IEEE Trans. on Radiation and Plasma Medical Science
|
Progress 06/01/18 to 05/31/19
Outputs
Target Audience:Undergraduate students: Our program untilized the Vertically Inegrated Projects course related to Plasma Medicine/ Agriculture with 15 students participating in research. Seminar: (Electrical Engineering Students) Presentation was given as a seminar to undergraduate students, junior level, and graduate students in electrical engineering on the plasma agriculture research topic and results. Seminar (Chemistry Students):Presentation was given as a seminar to students in chemistry and biochemistry on the plasma agriculture research topic. Local Industry: We met with the director of the Dairy West association which representsdairy food processors to discuss possible collaborations. Changes/Problems:The AC power supply used to drive the plasma soruce was a "resonant" supply. For arrays, this type of source does not work properly as it can not achieve high votlage (>4 kV). These power supplies are being modified to be non-resonant, and this change should improve performance of arrays. The thin (35 um) LTCC layer over the AC electrodes continues to have breakdown problems (pinholes). We have cheged the design to 110 um thick layer which has greatly reduced breakdown, but this increase in dielectric thickness does increase the oeprating votlage by ~20%. What opportunities for training and professional development has the project provided?The project has involved fourteen undergraduate students and one graduate student in a variety of training opportunities. Six undergraduate students were supported for research on this grant. Two additional students worked on this project leveraging Ralph Jones Fellowship support during 2018/2019. Their efforts included training on the fabrication, operation, and testing of the plasma sources. An additional eight students participated in the Vertically Integrated Projects (VIP) Plasma course. This course met for 1 credit hour in the fall of 2018 and 2 credit hours in the spring of 2019. Students in this course were trained on the testing and use of the plasma source, and were trained on the preparation of biofilms on a variety of solid substrate using cultures of foodborne pathogens. They exposed these samples to the plasma source, and then were trained to perform colony forming unit (CFU) course. In addition students: Prepared laboratory notebooks Were trained in giving oral presentations and then presented multiple times on their research in the course Presented posters at several undergraduate research conferences Wrote research reports Wrote Sigma Xi grants Dr. Browning presented the results of this work at the IEEE Conference on Plasma Science (June 2018). While at the conference he attended sessions on applications of plasma sources in industrial and agricultural environments. He also delivered a seminar on the topic during the Electrical and Computer Engineering Department seminar series. How have the results been disseminated to communities of interest?The results of our investigations have been presented at four conferences to a wide audience of professional, student, and community members. These conferences include: 2018 IEEE Conference on Plasma Science (Denver, CO, June 2018) 2018 Idaho Council on Undergraduate Research (Boise, ID, July 2018) 2018 Idaho INBRE Summer Research Conference (Moscow, ID, August 2018) 2019 BSU Undergraduate Research Conference (Boise, ID, April 2018) In addition, our work was presented to members of the public during the 2019 Bronco Days during tours of the research facilities conducted by our students. What do you plan to do during the next reporting period to accomplish the goals?As part of the next 6 months of the project, we expect to accomplish the following: Complete the development of a planar array of 9 plasma elements with embedded resistors and deploy the system in the biology experimental setup Use the planar array in biofilm experiments to determine the optimal operating parameters to kill bacteria including spacing and AC voltage Design and prototype a "radial" plasma array for use in pipes Demonstrate that the plasma arrays can ablate (sputter) biofilms when operated at high AC voltage Repeat biofilm kill experiments using the planar array for foodborne pathogens Present our results at the IEEE Conference on Plasma Science (June, 2019) Submit 3-4 journal articles on the plasma source preliminary results, on the existing foodborne pathogen results for different surface materials, on the array fabrication, and on new array biofilm exposure results.
Impacts
What was accomplished under these goals?
Foodborne pathogens create biofilms on surfaces encountered in food processing plants that are a significant source of contamination and threaten the food safety of the nation. These foodborne pathogens are ultimately responsible for millions of cases of disease every year, and the source of numerous product recalls that cost the agricultural industry millions in lost revenues. Reducing the incidence of foodborne pathogens contaminating foodstuffs during processing requires the use of harsh chemicals, and takes processing machinery off-line for a significant portion of the manufacturing cycle. A technology that could accomplish "in-line" decontamination of processing surfaces would save the agricultural sector millions of dollars in sanitation and food recall costs every year and reduce the incidence of foodborne illness.Cold Atmospheric pressure Plasmas (CAPs) use ionized gas to eradicate microbial pathogens on a variety of agriculturally important surfaces. In our project, we proposed to develop a device that would deliver plasma to a variety of food processing surfaces (steel, glass, plastic, rubber) and demonstrate its effectiveness in killing a variety of known foodborne pathogens (E. coli, Salmonella, Listeria monocytogenes, Staph. aureus, Pseudomonas, etc.). Our results have shown for all of the microbial pathogens that we have examined, that we could kill >90% of microbes found in biofilms on these surfaces with a CAP exposure of less than 5 seconds. These proof of concept results lay the ground work for developing a CAP device that could be deployed in a variety of food manufacturing environments (e.g. in-line on a food product conveyor belt) to eradicate pathogens and reduce the need for chemical cleaning. Deployment of such a system requires CAP sources capable of treating large areas. Our current research seeks to develop a proof-of-concept of a larger plasma array capable of treating a 2 cm x2 cm area at once. We have developed new sources which have been able to generate plasma over such an area, but we are still developing a usable array for demonstration of pathogen eradication experiments on a variety of surfaces. Such arrays could then be scaled to cover much larger areas for use in application in food processing (e.g., conveyor systems). Project goal: Develop a new type of Cold Atmospheric Pressure Plasma (CAP) sourcethat can be used in a variety of food processing settings for both treating food and cleaning food processing equipment. Objectives: to demonstrate that our system can deliver plasma reactive ions of sufficient flux to kill pathogens in a practical application a) Major activities completed / experiments conducted Several generations of CAP devices were fabricated with varying geometries and structures. A "standard" device was provided to the biology team to perform preliminary experiments on the efficacy in biofilm treatment. A second "engineering" system was developed and implemented using an existing data acquisition system and diagnostics. A new current transformer was acquired to measure the plasma discharge current; a new high voltage probe was acquired for the engineering system to measure the high voltage AC, and a new HV AC power supply was purchased for the engineering system. These diagnostics and support equipment were assembled into the new engineering system for CAP development. We have developed several generations of CAP devices with different geometries and interconnects to form these arrays. CAP array elements (individual sources) must have ballast resistors to ensure discharge uniformity. We have demonstrated acceptable discharge uniformity using external ballast resistors of ~100 kOhms. We have also begun development of internal ballast resistors fabricated using a thick film resistive paste. These resistors are integrated directly into the CAP structures, greatly decreasing the wiring requirements and making the array system far more compact. Preliminary results have shown proof-of-concept. Biology experiments have been conducted with the standard CAP source for treatment of foodborne pathogens on a variety of surfaces (glass, steel, plastic, and rubber) relevant in food processing. Biofilms were grown using a number of well-known foodborne pathogens, including Salmonella, E. coli O157:H7, and Listeria monocytogenes. The solid substrates containing pathogen biofilms were treated with the CAP source for a range of times using a mixture of argon and oxygen source gases. Following CAP exposure, the substrates were vortexed, and Colony Forming Unit (CFU) counts were performed to determine the best operating conditions for killing pathogens. b) Data collected Data collected includes plasma source Current-Voltage (IV) curves, plasma uniformity measurements with external ballast resistors, and internal ballast resistor measurements. Biological effectiveness studies include CFU counts vs. plasma exposure time for E. coli biofilms grown on a glass substrate. Our studies showed that biofilm bacteria were rapidly inactivated with plasma exposure (ED50=3 s) with a gas mixture of Ar and O2. c) Summary statistics and discussion of results The CAP discharge measurements were repeated 3 times for each turn-on/turn-off experiment. Results from the CFU count experiment represent the average of 3 independent experiments. These results demonstrate that we have successfully generated an ion flux capable of killing bacteria in biofilms. This result provides the basis for expansion of the studies as described in Objective 2. d) Key outcomes or other accomplishments realized In this project, we have built a new engineering system for CAP development to parallel the biology CAP treatment system used for actual pathogen treatment. A new discharge current measurement tool was integrated into the engineering system. CAP arrays with internal ballast resistors have been fabricated with 9 elements to cover a 2 cm x 2 cm area, but improvements in system planarity are still needed for improved plasma uniformity. Results have shown >90% reduction in CFU counts for E. coli on glass. 2. To demonstrate the effectiveness of our system on pathogens in a basic laboratory environment. a) Major activities completed / experiments conducted CAP development expanded to demonstration of proof-of-concept of a 9 element array using internal ballast resistors. Uniformity across the discharge as not achieved because of geometrical variation. Multiple discharge experiments were performed using a variety of interconnect configurations and structure geometries. Biological experiments included studies of a variety of pathogens ( E. coli, Salmonella, Listeria monocytogenes, Staph. aureus, Pseudomonas) on four different food processing related substrates: stainless steel, plastic, glass, and rubber. After plasma exposure, samples were vortexed, and CFU counts performed. b) Data collected CFU counts vs. plasma exposure time for Salmonella biofilms on stainless steel. Such experiments were repeated for the various pathogens:Salmonella, E. coli O157:H7, andListeria monocytogeneson stainless steel, glass, plastic, and rubber with CFU counts and determination of ED50. ?c) Summary statistics and discussion of results Each exposure experiment uses the average of 3 CFU measurements. These results clearly show that CAP sources can be used to kill a variety of bacteria biofilms on a variety of surfaces. While variation is observed, the results are generally very consistent. d) Key outcomes or other accomplishments realized The outcome of these experiments is the clear demonstration of the effectiveness of our plasma source in killing biofilm-associated pathogens on a variety of solid substrates that are encountered in a manufacturing/food processing environment. This accomplishment demonstrates the proof-of-concept for the use of CAP in killing biofilm in agricultural environments.
Publications
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2019
Citation:
IEEE Conference on Plasma Science:
PLASMA SOURCE FOR KILLING BACTERIA AND BIOFILMS ON SURFACES
Kate Benfield, Tiffany Berntsen, Daniel Moyer, Spencer Goering, Mariah Provost, Zeke Kennedy, Amanda White, Adam Croteau, Ryan Harper, Ken Cornell, Julia Oxford, and Jim Browning
Boise State University, 1910 W. University Drive
Boise, ID 83725 USA
Cold atmospheric pressure plasma (CAP) has been shown to kill or destroy bacteria and biofilm through reactive etch and sputter. Plasma can be used to debride wounds or to remove bacteria from food processing surfaces. including in food processing. Our parallel plate source operates at 20 kHz and 2-5 kV using Ar and O2 working gasses. The device is fabricated from a Low Temperature Co-Fired Ceramic (LTCC) and can be varied in discharge width from 2 mm to 5 cm. Embedded metal AC electrodes are buried 35 �m below the surface. The discharge gap is typically 0.5 mm. A HV probe and current transformer are used to measure the operating voltage and discharge current, respectively. The source has been used to kill bacteria on glass, stainless steel, rubber, and plastic with varying effectiveness. Colony Forming Unit (CFU) counts show reductions of 50 % in <10 s with 3-log reduction in 40-150 s depending upon surface. Biofilms tested so far include 2-day S. aureus (ATCC 25923), Listeria, Salmonella, and E-coli. The current source creates a single discharge line, so the samples must be rotated during exposure. Stacked arrays are being developed to allow full exposure of a large area. Embedded ballast resistors (100 k?) are being developed to provide discharge uniformity across an array of 9 sources. Finally, biofilms can be stained with Trypan blue to make them visible, and work is progressing on an optical imaging technique to identify the stained biofilm. These results will be presented.
________________________________
* This research is supported by the U.S. Department of Agriculture under the NIFA grant # 2018-67018-27881, by the National Institutes of Health (NIH) under Grant # 1R15EB024930-01A1. The project is also supported by the Leona M. and Harry B. Helmsley Charitable Trust and Boise State University College of Innovation and Design as a Vertically Integrated Projects course in Plasma Medicine.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
45th IEEE International Conference on Plasma Science (ICOPS 2018), Denver, CO, 6/24/18-6/28/18.
Benfield K, Bernsten T, Moyer D, Goering S, Provost M, Kennedy Z, Cornell KA, Oxford JT, Browning J*, Parallel plate cold atmospheric pressure plasma source for destroying bacteria and biofilms .
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
2018 8th Annual Idaho Conference on Undergraduate Research (ICUR), Boise State University, Boise, ID, 7/27/18-7/28/18.
Goering S*, Provost M*, Moyer D, McKenzie LJ*, Benfield K, Kennedy Z, McCarver J, Plumlee D, Oxford JR, Browning J, Cornell KA, A cold atmospheric plasma device to sanitize food industry surfaces .
McCarver J*, Kennedy Z*, White A*, Tenorio J*, Browning J, Plumlee D, Cornell KA Development of LTCC based cold atmospheric pressure devices for biofilm removal .
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
17th Annual Idaho INBRE Statewide Research Conference, Moscow, ID, 7/31/18-8/2/18.
Goering S*, Provost M, Moyer D, McKenzie LJ, Benfield K, Kennedy Z, McCarver J, Plumlee D, Oxford JR, Browning J, Cornell KA, A cold atmospheric plasma device to sanitize food industry surfaces .
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
16th Annual Undergraduate Research and Scholarship Conference (UGRC), Boise State Univ., Boise, ID, 4/15/19.
Provost M*, Goering S*, Neckels R*, Williams O*, Browning J, Cornell KA Plasma inactivation of biofilms produced by S. aureus and P. aeruginosa .
Provost M*, Goering S*, Ford Z*, Sullivan M*, Browning J, Cornell KA Plasma inactivation of biofilms on food preparation surfaces .
Goering S*, Nelson B*, Miller D*, Theel J*, Plumlee D, Oxford J, Browning J, Cornell KA A cold atmospheric plasma device to treat model wounds .
Tran S*, Sheets M*, Simmons Z*, Browning J Development and testing of a cold atmospheric pressure plasma array .
|