non-thermal fresh food sanitation by atmospheric pressure

46th International Conference on Environmental Systems ICES-2016-430 10-14 July 2016, Vienna, Austria

Non-Thermal Fresh Food Sanitation by Atmospheric

Pressure Plasma

Ross W. Remiker1, Robert J. Surdyk

2, and Robert C. Morrow

3

Orbital Technologies Corporation, Madison, WI, 53717

and

Magesh Thiyagajaran4

Glasram Technologies, Corpus Christi, TX, 78413

The Non-Thermal Sanitation by Atmospheric Pressure Plasma (NTSAPP) system was

developed for sanitizing fresh food on long-duration space missions. Plasmas are ionized

gases generated by electrical discharges and composed of electrons, positive/negative ions,

neutrons, and other neutral species. Plasma processes are known to be highly effective in

promoting oxidation, enhancing molecular dissociation, and producing free radicals and

other types of high energy phenomena. Non-thermal plasmas provide an effective way to

generate extremely reactive species and initiate a variety of chemical reactions of use in

surface sanitation of fresh fruit and vegetables. The NTSAPP system operates by passing a

carrier gas through plasma jet reactors. The generated plasma then flows with the air

through a rotating food sanitation chamber. After completing the sanitation cycle the carrier

gas flows through a catalytic scrubber to break down any harmful gas components prior to

being returned to the environment. Several prototypes were developed and tested, including

a system prototype that was designed to meet the interface requirements for ISS hardware.

The antimicrobial efficacy of the system prototype was tested with several process gases,

food items, and microbes. Testing showed that using bottled dry air and ambient lab air as

the process gas had similar results, and both performed better than other gaseous mixtures

that were tested. Lab air has the benefit of operation without pressurized gas lines and with

minimal consumables. Tests performed with samples of lettuce, tomato, and radish

inoculated with E. coli and Salmonella showed that fifteen minutes of sanitizing resulted in

reductions between 3.0 log10 and 3.8 log10. Testing with trained sensory analysis panelists

showed the process had no significant impact on taste, olfactory, or appearance quality.

Nomenclature

A = ampere

AFT = Applied Flow Technology

AP = atmospheric pressure

CFD = computational fluid dynamics

CFU = colony forming unit

DBD = dielectric barrier discharge

DC = direct current

DNA = deoxyribonucleic acid

EMI = electromagnetic interference

MLE = Middeck Locker Equivalent

NTSAPP = Non-Thermal Sanitation by Atmospheric Pressure Plasma

QDA = Quantitative Descriptive Analysis

1 Chief Engineer, Human Support Systems and Instrumentation, 1212 Fourier Dr., Madison, WI, 53717

2 Mechanical Engineer, Human Support Systems and Instrumentation, 1212 Fourier Dr., Madison, WI 53717

3 Chief Scientist, Human Support Systems and Instrumentation, 1212 Fourier Dr., Madison, WI 53717

4 Chief Technology Officer, 4122 Pontchartrain Dr., Corpus Christi, TX 78413.

International Conference on Environmental Systems

2

psig = pounds per square inch, gauge

ROS = reactive oxidizing species

RPM = revolutions per minutes

SBIR = Small Business Innovation Research

UV = ultraviolet

VDC = volts, direct current

W = watt

I. Introduction

HIS Non-Thermal Sanitation by Atmospheric Pressure Plasma (NTSAPP) technology utilizes non-thermal,

atmospheric pressure (AP) plasma, generated by high-energy electrical discharge at ambient temperature and

pressure. Plasmas are ionized gases that can be generated by electrical discharges and are composed of electrons,

positive/negative ions, neutrons, and other neutral species. The goal of this technology is to sanitize fresh fruits and

vegetables on long-duration spaceflight missions with minimal impact on food quality.

In addition to prepackaged menu items, food systems on long-duration missions would include: (1) the

production of some fresh salad crops grown on board and (2) production of food items from scratch components

packaged in bulk.1 Possible scenarios selected from the ALS Reference Missions Document (JSC-39502), including

Mars Transit, Mars Surface Habitat Lander, and Evolved Mars Base, call for post-harvest procedures or

technologies to aid in providing acceptable, safe, and nutritious salad crops and food preservation technologies to

preserve ingredients processed in situ.

Although it has not been determined how much sanitation will be required for hydroponically grown fresh fruits

and vegetables in an enclosed environment, the plants themselves in such a growing system might facilitate the

development of pathogenic organisms. Seed borne inoculum for human pathogens has recently been recognized as a

factor for food borne illness outbreaks. Pathogenic bacteria can infiltrate cracks, crevices and intercellular spaces of

seeds.2 In addition, potentially pathogenic human-associated bacteria might survive in the rhizosphere of plants

grown on orbit.3,4

Some of these opportunistic organisms could pose a threat to crew health given the depressed

immunity of humans in a space environment.

Non-thermal AP plasma is an effective disinfection and decontamination technology, due to high removal

efficiency, energy yields, and low cost.5 Plasma processes are well known to be highly effective in promoting

oxidation, enhancing molecular dissociation, and producing free radicals and other types of high energy phenomena

(e.g., UV radiation and shock waves) to enhance chemical reactions.6 The special properties of non-thermal plasmas,

especially those produced at ambient pressure without the need for vacuum, provide a great way to generate

extremely reactive species and initiate a variety of chemical reactions—all at ambient temperature and pressure.

The NTSAPP technology supports sanitation of food preparation surfaces and equipment, food storage

containers, surface sanitation of delivered fresh food, freshly grown fruit and vegetables, and freshly prepared foods

in a space-based habitat. This technology can function in reduced gravity and pressure environments, and is efficient

in terms of mass, power, volume, and expendable resource (e.g., water) use, with minimal byproducts.

Various non-thermal plasma techniques have been studied for disinfection of food related products including

packaging materials, medical devices, and other substrates. It has been shown that non-thermal plasmas are highly

effective in sterilizing plastics, medical devices, and certain fresh produce.7

Several different mechanisms are involved in non-thermal plasma sanitation. To kill or inactivate

microorganisms by most of the chemical/physical methods, it is essentially required to target the microbial

cytoplasmic membrane and compromise its structural and/or functional integrity. Studies have shown that factors

contributing to the activity of chemical disinfection include the constant of diffusion in the microorganism, the

electric charge of the disinfectant, and the pH of the medium. The most essential factor, however, is the oxidizing

power of the disinfectant, which happens to be one of the key characteristics of the plasma state.8 In addition,

plasma may have a general mechanical effect on the surface of the living microorganism as a result of the intense

bombardment from hydroxyl and other heavier free radicals, which cause surface lesions that the living cells cannot

repair sufficiently quickly, leading to cell destruction.9 Another phenomenon, called electroporation, occurring

under a pulsed electric field and causing destruction of microorganisms, also exists in the plasma environment.

Detailed studies10

have shown that exposure to plasma results in perforations in the membranes of microorganisms,

which in turn lead to increased membrane permeability that affects the transmembrane potential of the cells and their

ability to regulate intracellular pH.11

Orbital Technologies Corporation (ORBITEC) leveraged their experience developing plasma technologies for

surface property modification, water decontamination, and air decontamination to develop plasma food sanitation

T


3

technology under SBIR Phase 1 and 2 contracts through NASA JSC. The prototypes developed under these SBIR

contracts include breadboard power supplies, stand alone plasma reactors, subscale plasma chambers, and an

integrated system prototype. This paper describes the hardware development and testing that was performed under

these SBIR contracts.

II. Summary of NTSAPP Early Developments

Early ORBITEC NTSAPP prototype designs built upon many previous ORBITEC plasma technology

developments, including plasma surface modification systems, plasma water decontamination systems, and plasma

air decontamination systems. In addition, prototype plasma generating power supplies, reactors, and chambers were

developed and tested in the Phase 1 SBIR development and early in the Phase 2 SBIR development.

A. Summary of Early Designs

Plasmas can be generated easily under reduced atmospheric pressure, but to initiate and maintain steady

discharges at ambient pressure imposes many limitations on reactor design. The most limiting factor in food

processing applications using atmospheric pressure plasmas is the small volume of the reactive zone. Discharges

occur around electrodes for AP plasma reactor designs and do not extend very far from the electrode surface, which

means it is easy to treat flat surfaces but difficult to process objects with complicated shapes. Though reactive

oxidizing species (ROS) generated by plasma can survive and be carried to a remote location for sanitation

treatment, their lifetime is limited and the quantity is significantly lower than that found within the discharge zone,

resulting in less effective sanitation at the remote location. It was the main objective of this development effort to

identify an AP plasma design that provided the largest volume of the reactive zone, which meant more flexibility

during application, and yet remained effective against microorganisms.

1. Power supply

The NTSAPP power supply provides the electrical input to the plasma reactor. It comprises three sections that

work together to generate a high voltage pulse used by the reactor to create plasma. First, control electronics

generate a series of pulses with precisely controlled frequency and width. These pulses are optically coupled to a

driver module, which converts the control pulses to high current pulses. Finally, the high current pulses are sent to

the primary winding of an ignition coil (transformer), storing energy in a magnetic field. When the pulse drops from

high to low, the energy stored in the magnetic field is released through a secondary coil, creating a high voltage

surge. This surge is routed to the reactor, where it creates plasma used for decontamination.

2. Plasma reactor

The reactor consists of two coaxial electrodes separated by dielectric materials. The central electrode is powered

and the process gas flows past its central electrode and exits the device. Dielectric barrier discharges are produced at

the exit and a plasma jet/plume forms near the exit due to the flow of the process gas. The plasma discharge from an

early reactor design developed during Phase 1 is shown in Figure 1 (left).

Several reactor designs were analyzed and tested to evaluate performance of individual reactors before

incorporating them into plasma chambers. Reactor designs were analyzed with computation fluid dynamics (CFD)

software and with COMSOL Multiphysics Software – Plasma Module to optimize performance by simulating

different reactor geometries, gases, and gas flow rates.

3. Plasma chamber

Early prototype plasma chambers incorporate one or more reactors flowing plasma and plasma reaction products

into a single volume. The center image in Figure 1 shows four individual jet reactors incorporated into a single

reaction chamber. ORBITEC performed CFD analysis on the gas flow through the reactor to modify the design as

needed to optimize flow through the chamber. Figure 1 (right) shows a single reactor that flows into plasma chamber

with a cross section similar to the chamber in the integrated system prototype.


4

Figure 1. Early plasma reactor and prototype chamber designs: plasma reactor in operation (left);

four reactors mounted to a single chamber (center); single reactor mounted to a single chamber (right).

B. Summary of Previous Testing

These earlier investigations included evaluations of electrical power supply variables such as voltage, pulse

width, and pulse frequency, along with many reactor design variables, including geometry, materials, and

construction methods.

Different process gases, gas flow rates, and gas flow methods (recirculated vs. single-pass flow) were tested, but

when selecting a process gas and gas flow method there is more to consider than strictly antimicrobial performance.

Operating with lab air (or cabin air) has a major mass advantage over any pressurized bottled gas. A fresh supply of

bottled gas would require a pressurized tank and pressure regulator that add mass to the system. In addition the

consumable gas would need to be resupplied as it is used during decontamination operations. A lab air (or cabin air)

system would require no pressurized hardware and would have no consumables. In addition, any bottled gas that

contains oxygen generates ozone, which is also the case for lab air, so an ozone scrubber would be required. Single-

pass process gas operation would require continuous ozone scrubbing after the gas passes through the system. A

recirculating gas system would only need to scrub ozone after the sanitation operation is complete and the system air

is vented back to cabin, and may be able to operate safely with a smaller ozone scrubber. If an oxygen-free bottled

process gas is used, ozone would not be generated, and there would be no need for an ozone scrubber.

1. General plasma and thermal testing

Preliminary operational testing was conducted on early prototypes to validate that arcing was not present

between reactors and there was no arcing from reactors to the test samples in the chamber. Additional testing was

performed to confirm that the temperature of the plasma did not raise the temperature of the samples during testing.

The temperature of the gas within the chamber volumes was approximately 5°C to 6°C above ambient temperature,

which indicated that food quality would not likely be affected.

Optical emission spectra of the plasma discharge generated by the prototype shown on the right in Figure 1 were

measured. This was done to identify the various reactive plasma species and to determine the plasma temperature.

Optical emission spectra testing was performed by Glasram Technologies, a commercial spinoff of the Plasma

Engineering Research Laboratory (PERL) at Texas A&M University – Corpus Christi. Dr. Magesh Thiyagarajan is

the founder and Chief Technology Officer of Glasram Technologies and is also Assistant Professor of Engineering

at Texas A&M University – Corpus Christi and director of PERL. Dr. Thiyagajaran oversaw all analysis and testing

performed by Glasram Technologies. The main output of this testing is the plasma intensity, which is measured in

photon counts and demonstrates the identification of species needed for microbial inactivation and their relative

intensities. The plasma emission intensity in conjunction with plasma density was taken into consideration for

diagnosing the effectiveness of different experimental configurations. Process gases of argon, dry air, lab air, and a

mixture of 99% argon and 1% oxygen were used for these tests. All gases were tested with a single pass of gas

flowing through the system and with gas recirculated through the system. Plasma performance with argon and the

argon/oxygen mixture performed better than bottled dry air and lab air. Recirculated lab air did not perform quite as

well as fresh bottled air, but it showed potential for good microbial inactivation. In general, single-pass process gas

performed better than recirculated process gas.


5

2. Antimicrobial testing

The antimicrobial efficacy of the plasma chamber shown in the center of Figure 1 was tested at the Food

Research Institute at the University of Wisconsin. Professor Amy Wong of the UW Department of Bacteriology

oversaw all test activities. Tests were performed on the plasma jet reaction chamber to evaluate antimicrobial

performance under different conditions. Test substrates included Teflon disks, Teflon balls, lettuce, and tomatoes.

All samples were inoculated with three strains of Salmonella and antimicrobial performance was tested with argon,

dry air, nitrogen, and lab air, at two different flow rates. Two different functional designs were tested; a single

reactor that treats surfaces in direct contact with the plasma plume, and a plasma reaction chamber that incorporates

four jet reactors and treats three dimensional surfaces within the chamber volume. The plasma chamber had limited

success with sanitizing Teflon samples, but was quite successful sanitizing lettuce and tomato samples. Test results

were very encouraging, but generally inconsistent, especially in terms of optimizing process gas and operating

parameters.

III. NTSAPP Integrated System Prototype Development

The knowledge gained from previous plasma developments, early plasma food sanitation prototypes, and

ORBITEC’s previous ISS payload developments went into the design and testing of the NTSAPP integrated system

prototype. Early benchtop prototypes had the power supply electronics, process gas delivery system, and

reactor/chamber as separate, interconnected hardware items. The integrated system prototype packages those

individual subsystems, along with additional functionality and safety controls, into a single enclosure that was

designed to interface with an ISS EXPRESS Rack single Middeck Locker Equivalent volume and utilize the 28

VDC, 20A power connection. The system prototype is a rear breather that is designed to interface with the

EXPRESS Rack avionics air for cooling. Avionics air flow through the integrated prototype internal volume is

driven by a single axial fan within the prototype. Overall dimensions of the system are 24.6 inches x 18.39 inches x

10.75 inches, with a weight of 66 lb. The power draw during plasma processing is approximately 68W.

A. Design

The system functional schematic is shown in Figure 2. The main components of the system are the plasma

reactors, processing chamber, process gas pumps, catalyst bed, high voltage power supply, and control boards. At

the time the system was designed we expected best performance from a pressurized gas source, and if a pressurized

gas source was used, then recirculation of the process gas would be preferred to minimize consumables. So this

design was developed primarily for recirculating flow from a pressurized gas source. Other process gases and flow

conditions could work with this hardware design, but there would be limitations in ozone scrubbing capability.

In recirculation mode four parallel pumps pressurize an orifice manifold, which contains orifices to provide

equal gas flow to five plasma reactors. Note - the four parallel pump configuration provides the desired pressure and

flow rate with lower mass and volume than any single pump that was available. The plasma generated by the five

reactors flows through the processing chamber that houses a rotating basket that contains the specimens to be

sanitized. After the process gas leaves the processing chamber, it is either recirculated through the system by the

pumps, or vented outside the unit, depending on the operating mode and stage of the decontamination process.

To control the voltage pulse to the reactors the driver signal from the control board will be supplied to the power

supply board. The driver signal defines the frequency and pulse width of the reactor supply voltage. The power

supply boards supply the spark coil with a low voltage signal where the spark coil transforms low voltage to high

voltage. The high voltage signal is then supplied to the plasma reactor.

The NTSAPP internal enclosure is segmented into two compartments to isolate the control board, pump, motor,

and sensors from high-voltage electronics. The high-voltage compartment contains the power supply boards, power

converter assembly, spark generators, and processing chamber assembly. The two volumes are separated by

aluminum walls to prevent EMI that is generated by the high-voltage components from interfering with the low-

voltage signals in the control electronics. Signals between the control board and power supply board are transmitted

through fiber optic cables. Power wiring that runs from the control volume into the high-voltage volume have inline

EMI filters to reduce EMI transmission into the control volume. Cooling avionics air flow is shared between the

volumes with air flowing through a perforated EMI screen. Gas lines bridging the high and low voltage sections

utilize dielectric connectors mounted on the partition panel. The outer enclosure is aluminum with a conductive

chromate conversion coating for grounding and EMI protection.


6

Figure 2. NTSAPP system prototype functional schematic.

In operation, the food specimens are placed in a carrier basket which is inserted into the processing chamber

through a door located on the front panel of the enclosure. Figure 3 shows the chamber with five reactors mounted to

it, and shows how the basket is installed into the chamber through the front panel. The basket is magnetically linked

to the drive motor that rotates the basket at a low rotational speed of approximately 3 RPM. The basket rotates

because with a static chamber, it would be possible for air flow through the chamber to force all food items to one

location near the chamber air outlet, and possibly limit exposure of the food to the reactive plasma products. A

rotating chamber provides a mild tumbling action that ensures that all surfaces of the food are contacted by plasma

and plasma reaction products.

Figure 3. Five plasma reactors mounted to chamber housing (left), carrier basket installation (right).

Processing Chamber

T2

P2

Drive Motor

24/13.5 VDC

Converter

Control Board

NTSAPP Enclosure

Process Gas

Fill Line

Vent Valve

Fill Valve

Chamber Door

Limit Switch

Process Gas Line

Pump

Power

Supply

Board

Spark

Coil

Orifice Plasma Reactor

DP

P1 T1

Orifice

Manifold

Vent

Line

Filter 40 micron

Regulator

5 PSI

Filter

60 micron

Check Valve

O3 Catalyst Bed

Plasma Reactor

Plasma Reactor

Plasma Reactor

Plasma Reactor

Spark

Coil

Spark

Coil

Spark

Coil

Spark

Coil

Orifice

Orifice

Orifice

Orifice

Power

Supply

Board

Power

Supply

Board

Power

Supply

Board

Power

Supply

Board


7

The assembled NTSAPP system prototype is shown in Figure 4. The front panel includes all user control

buttons, liquid crystal display (LCD), power connector, data connector, power switch, circuit breaker, chamber

access door, purge vent, and process gas quick disconnect interface. The user is able to control all functionality of

the system with four buttons, and all system options are displayed on the LCD. The gas supply quick disconnect is

for connection to an external high pressure gas source. After plasma processing the process gas vents through the

external filtered gas port on the front panel.

Figure 4. NTSAPP system prototype

B. System Operation

The prototype can support different operational modes, including single-pass flow from a pressurized gas source,

recirculated flow from a pressurized gas source, and recirculated flow from an unpressurized gas source (lab air).

1. Recirculated pressurized gas

Plasma decontamination from a pressurized bottled gas source is completed in three main processing phases: (1)

system gas purge, (2) plasma processing, and (3) ozone removal.

System Gas Purge - The initial step in the processing procedure is a complete purge of all gas within the

processing chamber and system gas lines. The purge replaces all gas within the system with gas from a process gas

tank. The gas from the high-pressure tank is injected into the system through a filter, inlet solenoid valve, and a

pressure regulator that reduces pressure to 5 psig as it enters the system process lines. Four diaphragm pumps are

activated to circulate the purge gas throughout the system. The purge gas flows for a predetermined amount of time

until the injected process gas within the system is close to a 100% concentration. As the gas flows through the

system it exits through a venting solenoid valve just upstream of the pumps and out through the front panel vent.

Plasma Processing - Once the gas purge is complete the system closes the inlet and vent solenoid valves and the

four diaphragm pumps continue to circulate gas throughout the system. The gas then passes through the plasma

reactors into the chamber where the food resides inside the slowly rotating basket. Then gas exits the chamber and

flows back to the pumps. This cycle continues until the desired testing time is complete.

Ozone Removal – If the process gas contains oxygen, ozone will be created during the plasma generation

process. The purpose of this step is to eliminate any ozone from the process gas before mixing the process gas with

the outside environment. During this step all reactors are shut off and a three-way valve diverts the airflow through a

catalyst bed. This breaks down any ozone that may be remaining within the system. The catalyst bed is sized for

single pass removal efficiency near 100%. The required ozone removal treatment time was experimentally

determined with the integrated system prototype under actual processing conditions. A safety factor is applied to the

required removal time to ensure safe operation.


8

2. Single-pass flow pressurized gas

It is possible to operate the NTSAPP prototype with a continuous single-pass flow with bottled gas, however this

method requires more consumable gas than using recirculated process gas, and this prototype design does not have

the functionality to continuously scrub the ozone that may be generated and expelled through the front panel vent

during processing. Single-pass flow has three phases of operation, similar to operation with recirculated bottled gas.

System Gas Purge - The initial gas purge is not as critical for single-pass flow as it is for recirculated flow. The

purge phase is similar to that with recirculated gas, but the duration is shorter in single-pass flow mode.

Plasma Processing - During the plasma processing phase, the pumps circulate gas through the system while fresh

gas continuously flows into the system from the pressurized source and process gas exits through the front panel

vent.

Ozone Removal – During the ozone removal phase the inlet and vent solenoid valves are closed and gas is

circulated through the catalyst bed. This phase is the same for recirculated mode and single-pass flow mode.

3. Lab air

The prototype has the same internal operations for lab air and pressurized gas, but the effect on the process gas is

different because there is no pressure forcing process gas from the inlet to the outlet.

System Gas Purge – The purge phase is not very effective without pressurized gas flowing into the system, but

this phase also isn’t essential because the gas inside the unit is essentially the same as lab air.

Plasma Processing – Plasma processing in recirculating mode is the same for lab air as for a pressurized gas, but

there is no equivalent of “single-pass flow” of lab air through the system. With the vent valve open, there is

effectively a continuous leak of process gas to the outside, rather than true flow from the inlet to the outlet. Rather

than refer to the different modes and “recirculating” and “single-pass”, more accurate terminology is “closed

recirculation” and “vented recirculation” The next generation design would include more capability for true single-

pass flow of lab air during plasma processing.

Ozone Removal – The gas inlet is closed during the ozone removal phase so the effect is the same whether the

process gas is pressurized or lab air.

C. Analysis

Analyses were performed on the specimen chamber, process gas system, and the prototype internal cooling air

flow volume to ensure that the system would operate as desired.

1. Flow through specimen chamber

A CFD analysis was conducted to evaluate the flow profile of the gas from the reactors through the chamber and

basket. No food was incorporated in this analysis because the location and shape of the food would essentially be

random and would further complicate the analysis. The analyses were conducted at a number of different gas flow

rates encompassing the range of flow rates that may be used in operation. As expected, the lowest flow rates had the

most consistent flow throughout the chamber volume, but also showed areas of very little mixing within the volume.

The highest flow rates showed good air mixing within the chamber, and the greatest variation in flow throughout the

volume.

2. Flow through process gas system

A fluid analysis was conducted on the pumps and piping system to specify the appropriate orifice size to deliver

the desired flow of gas for each reactor with the bank of pumps chosen for the system. The set of analyses were

conducted with AFT Arrow 4 piping network analysis software. The piping network was generated within AFT

Arrow 4 and all relative valve data along with reactor and filter flow parameters were incorporated in the software.

Results of this analysis led to selection and sizing of the orifices upstream of the reactors.

3. Flow through system enclosure

A flow and thermal CFD analysis of the complete system was conducted to verify that the flow through the

enclosure was sufficient to maintain electronics at safe operating temperatures. The system is cooled by a single fan

pulling air from the rear avionics into the enclosure then exiting through the rear avionics vent. Two additional fans

reside on the DC-DC converter heat sink assembly and circulate internal cabin air.

This is a conservative method to analyze the system’s thermal capability because it assumes the hardware rejects

100% of its power into heating the air inside the unit and also assumes the hardware is running continuously at

steady-state conditions. The system will actually be run for only short durations, approximately 10-20 minutes.

The results from the thermal analysis show that even in the worst case the temperature of the equipment remains

within allowable temperature limits. The highest temperature resides within the three-way solenoid valve; this is not

of concern since it will be powered for only approximately one minute each time food is processed.


9

IV. NTSAPP Integrated System Prototype Testing

The NTSAPP integrated system prototype was tested to evaluate plasma generation, antimicrobial efficacy,

ozone output, and effect on food quality.

A. Plasma Testing

Glasram Technologies conducted several tests to characterize and optimize the system’s plasma field. Testing

metrics included: plasma density, temperature, ionic species, and reactive agents. The primary purpose of the

plasma testing was to narrow down the number of operational parameters that would be varied in the antimicrobial

testing. The following process parameters were varied during testing: process gas type (dry air, lab air, argon, and

argon with 2% oxygen mix) and process mode (single-pass flow of fresh process gas and recirculation of process

gas). The process gas flow rate and high voltage pulse frequency were set to values that were optimized during

previous testing with earlier prototypes.

Optical emission spectra of the discharge were measured to identify various reactive plasma species and to

determine the density, gas composition, and plasma temperature. The electron density was calculated by the line

broadening of the respective spectral emissions. The ozone concentration was measured within the plasma chamber.

For this series of tests, the standard front panel door was replaced with a door that was designed specifically to allow

all of the necessary sensor access. A test was performed for each process gas and process mode combination.

The optimization testing performed on the NTSAPP system prototype showed that single-pass flow of argon and

Ar/O2 had higher plasma intensity and plasma density than other gases and flow modes, and had lower ozone

concentration within the chamber. The highest ozone concentration measured within the chamber for all tests was

0.06 ppm with fresh lab air. The plasma intensity and density of bottled dry air and lab air were not as high as Ar

and Ar/O2, but they have some major advantages in terms of operations and system complexity compared with other

process gases, so they were not ruled out at this time. Based on the plasma test results, we decided to move forward

with microbial testing of all four gases and both flow configurations (fresh flow and recirculation), with plans to

narrow down our gas and flow configuration choice early in microbial testing.

B. Antimicrobial Testing

Lettuce, tomato, and radish were selected for testing that involved inoculation followed by low temperature

plasma treatment. Lettuce was selected due to it being a primary salad crop and at the time was scheduled to be

flown on the ISS as part of the VEGGIE Vegetable Production System. Lettuce has convoluted leaves with smooth

surfaces. Lettuce is also a good analog for candidate salad crops such as spinach, bok choy, chard, cabbage, and

other leafy greens. The second candidate test crop, tomato, is a desirable crop for use on orbit and can be a relatively

prolific producer. It is also a reasonable analog for peppers and snap peas (edible pods), having a similar smooth,

somewhat glossy surface. The third candidate test crop, radish, is a popular vegetable for space applications as it has

a spiciness that is desirable in a microgravity environment where taste is diminished due to fluid shifts to the upper

body and head. Radish is also a reasonably good analog for the candidate salad crops beet, carrot, onion, and celery

as the edible part grows near the soil surface and is more susceptible to contamination. It has a rough texture similar

to beet and carrot, and often has uneven surface characteristics such as cracks or protrusions, and thus provides a

sanitation challenge for the NTSAPP system.

Vegetable samples were inoculated with nonpathogenic strains E. coli and Salmonella typhimurium. All tests

were run with nine samples at a time. For each set of variables, three identical tests were run with plasma generation

and three identical control tests were run with all parameters (process gas, flow rate) the same as the corresponding

plasma tests, but with no plasma generation. For a general reference, the control runs yielded microbial counts of

approximately 2 x 106 for lettuce, 3 x 10

5 for tomato, and 3 x 10

6 for radish. The log reductions from processing are

the comparisons between the three test runs and three control runs for each set of parameters.

Combinations of the following parameters were evaluated:

Process gas: lab air, dry (bottled) air, argon, argon/oxygen (98% argon/ 2% oxygen)

Gas flow condition: single-pass flow, recirculation

Inoculum: Salmonella (non-pathogenic strain), E. coli (non-pathogenic strain)

Food sample: lettuce, cherry tomato, radish

Process duration

Sample containment: tumbling loose in basket (the intended method used in operation); contained in

sample isolation tray (to maintain constant location within process gas flow)


10

1. Fixed position within rotating chamber

The first series of antimicrobial tests evaluated various

system parameters using a sample containment tray

installed in the rotating basket. This was done to evaluate

the microbial kill performance at different locations

throughout the entire plasma processing chamber volume,

and to determine if the antimicrobial performance was

consistent throughout the entire chamber volume. These

tests would show if there is a performance difference near

the outside of the chamber, compared to the center of the

chamber, and if location relative to the front or back of the

chamber results in different performance. The positions

within the sample containment tray are shown in Figure 5.

Each trial used nine samples of lettuce treated with E. coli.

Three trials were run with plasma at each condition, and

three control trials were run at each condition. The average

resultant CFU for each plasma trial was calculated, along

with the average CFU of each control group of three repeated trials. These average values were used to calculate the

average CFU log10 reduction at each condition. The reduction is calculated as the difference between the treated

samples and the control samples after processing.

This trial set was conducted using multiple process flow conditions, process gases, and process times. The data

showed the following:

There is no significant variation in performance throughout the sample containment volume.

Lab air and bottled air performed better than argon and the Ar/O2 mixture

Lab air and bottled air had very similar performance

Microbial reduction with recirculated lab air after 10 minutes of treatment was approximately 2 log10

reduction, compared with 1 log10 reduction after 5 minutes.

Based on results of the fixed-position tests, we removed the argon/oxygen mixture from process gas

consideration, since it did not perform as well as dry air or lab air. Argon also did not perform as well as air, but has

the advantage that there is no ozone produced, so we chose to perform initial tumbling tests with argon, with both

recirculating and single-pass flow.

There was no significant difference in antimicrobial performance between lab and bottled dry air, and lab air has

significant operational advantages to bottled air, so bottled air was removed from future consideration.

2. Free position (tumbling) within rotating chamber

Slow tumbling is the intended processing method for NTSAPP operation so the majority of antimicrobial tests

were performed with samples tumbling freely in the chamber. For all tests, nine food specimens were tumbled in the

chamber at once. For each set of parameters, three plasma tests and three control tests were run. The control tests

were run identically to the plasma tests, except with no plasma generation.

Initial tests were performed with argon as the process gas, with lettuce samples inoculated with E. coli. Results

showed that performance with argon was not effective enough to warrant further consideration. The remaining tests

were run with lab air and all possible combinations of the following test variables:

Gas flow mode – true recirculation and vented recirculation

Food specimen – lettuce, tomato, and radish

Inoculate – non-pathogenic strains of E. coli and Salmonella

Process time – 10 minutes and 15 minutes

As noted previously, the system prototype was designed with the expectation of using a pressurized gas in

recirculation mode. This is because early tests indicated that bottled gases performed better than lab air, and

recurculating the gas within the system would conserve consumables. Operation with lab air will work the same as

with pressurized gases for recirculation mode, but there will be no true single-pass mode. Since lab air does not have

a pressurized source to drive it through the system, there will not be true fresh flow of lab air through the system, but

rather a continuous leak of process gas through the vent valve. One disadvantage of the vented mode of operation is

that there is not continuous ozone scrubbing of the process gas before it exits the system. With potential for ozone

leaking out of the system during vented mode, all tests were run with the system prototype in a fume hood. Note that

Figure 5. Fixed sample locations within

basket


11

any future versions of this design will include pumps to draw in lab air and move it through the system and ozone

scrubbing on the vent line to mitigate the ozone concern.

Similar to the fixed position tests, nine food samples were processed per trial. For each combination of variables,

three trials were run with plasma and three control samples were run with no plasma generation. The average

resultant CFU after each plasma trial was calculated, along with the average resultant CFU of each control group of

three repeated trials. These average values were used to calculate the average CFU log10 reduction at each condition.

The reduction is calculated as the difference between the plasma treated samples after processing and the control

samples after processing. A summary of the results of the antimicrobial tests are shown in Table 1.

Table 1. Summary of NTSAPP antimicrobial performance with lab air as process gas

In general the lettuce and tomato samples respond similarly to treatment. The radish seems to resist the

antimicrobial treatment more than the other food types. This decrease in microbial reduction is likely due to the

surface properties of the radish. The radish surface morphologies include hair-like roots and structures on the

surface, as well as microscopic cracks. Radish has presented similar difficulties with other antimicrobial systems

such as disinfecting wipes.12,13

To account for the challenging surface, radish must be processed longer than foods

with smooth surfaces to achieve the same microbial reduction.

C. Ozone Generation

The use of any process gas that contains oxygen generates ozone during this plasma process. The production of

ozone internal to the system is beneficial, as it is an effective antimicrobial agent. However, ozone that escapes the

system to the surrounding atmosphere could be potentially hazardous to system operators.

The ozone level outside the unit was monitored from approximately two feet away during operation. For these

ozone evaluation tests the unit was operated in a normal lab environment, not in a fume hood. Ozone measurements

during the trials indicated ozone concentration of 0.2 to 0.4 ppm, when operated in the recirculated mode, and 0.8 to

1.5 ppm in the vented mode.

There is no maximum ozone concentration listed in JSC 20584 – Spacecraft Maximum Allowable

Concentrations for Airborne Contaminants, but ideally there would be no measurable ozone in any spacecraft

environment. The Occupational Safety and Health Administration’s ozone concentration safety limit is 0.1 ppm. The

measured ozone concentrations during these tests exceed this limit. This issue will be addressed in future designs.

The most important future design change is addition of a catalytic ozone scrubber to the vent line. Additional

solutions are readily available to reduce ozone leakage outside the system, including welded tubing connections and

using better sealing materials to reduce leakage. These features will be incorporated into all future developments.

D. Food Quality Testing

The effect of NTSAPP processing on food quality was tested with the University of Wisconsin Sensory Analysis

Laboratory by trained food quality test panelists.14

Romaine lettuce, cherry tomato, and European radish were tested.

Each vegetable product was sampled and separated into two groups: a control group, and a treatment group that was

further treated in the NTSAPP system prototype for 10 minutes. All samples were purchased in a local supermarket

and tempered for an hour to 22.0°C in an incubator before evaluation.

Food Sample Mode

Duration

(minutes)

E. coli reduction

(log10)

Salmonella reduction

(log10)

Lettuce Recirculated 10 2.42 2.28

Lettuce Vented 10 3.05 2.91

Lettuce Recirculated 15 2.83 2.64

Lettuce Vented 15 3.63 3.53

Tomato Recirculated 10 2.54 2.31

Tomato Vented 10 3.12 2.96

Tomato Recirculated 15 2.93 2.70

Tomato Vented 15 3.81 3.66

Radish Recirculated 10 1.85 1.62

Radish Vented 10 2.63 2.46

Radish Recirculated 15 2.19 2.00

Radish Vented 15 3.18 2.96


12

Lettuce heads were sampled by cutting leaves out of the head, rinsing them in running cold water, cutting the

upper 10 cm of each head, and removing the stem portion to control variability throughout the leaf as much as

possible. The cuttings were then randomly separated into the control and treatment groups. Cherry tomatoes had

leaves removed, and were rinsed in cold, running water, and separated into the control and treatment groups.

Radishes had their root and stem removed by cutting, were rinsed in cold, running water to remove residual dirt, and

separated into the control and treatment groups.

Descriptive evaluation was carried out using Quantitative Descriptive Analysis (QDA) by 10 panelists with at

least 40 hours of training on the QDA technique and 12 hours of training on the specific products evaluated. All

samples were evaluated in quadruplicate. All samples were washed before processing, and were not inoculated.

Samples were processed about two hours before the start of the testing and were stored with control (untreated)

samples in a controlled temperature incubator prior to testing.

Test safety was addressed through development of a food safety analysis of the NTSAPP system prototype

hardware followed by submitting a test plan to the NASA JSC Human Test Subject Internal Review Board for

approval. The University of Wisconsin Sensory Analysis Panel has developed procedures for handling and

processing food test samples for many years and these were applied to procurement, preparation, NTSAPP

treatment, and subsequent post-test handling of the NTSAPP test samples.

Statistically significant differences were found in the sweetness of radish – the treated samples were found to be

slightly sweeter than the controls. However this difference was not great enough to be considered a

biological/functional difference. This is probably due to natural sample variation and not the treatment. Panelists

commented that the treated lettuce samples had a faint ‘dampness’ aroma reminiscent of standing near a lake, and a

few panelists noticed a slightly lower crispness in the center of treated radishes, but the results found no significant

differences in the overall crispness. Both of these observations could be due to natural sample variation and are not

indicative of changes caused by the treatment of the samples. Overall, no practical differences were found between

the treated and control samples.

V. Conclusion

Non-Thermal Sanitation by Atmospheric Pressure Plasma technology has been shown, through a series of

prototype developments, to have potential for successfully sanitizing fresh foods in space with minimal consumables

and no significant impact on food quality. The prototypes that were developed include an integrated system

prototype that was packaged to fit into a single EXPRESS Rack Middeck Locker Equivalent (MLE). The system

prototype was designed with the intention of operating with the process gas recirculating through the system to

conserve consumable gas, but is also capable of operating with pressurized gas flowing though the system in a single

pass or with the system vented to lab air, but the prototype design does not provide optimal ozone scrubbing in these

single-pass or vented modes. Antimicrobial efficacy was tested with a number of bottled pressurized gases and with

lab air as the process gas. Based on early test results lab air was selected as the preferred process gas because it has

similar performance to bottled pressurized gas and it provides a significant mass advantage due to the absence of a

pressurized subsystem and consumable process gases. Tests performed with a vented, recirculated lab air on samples

of lettuce, tomato, and radish inoculated with non-pathogenic strains of E. coli and Salmonella showed the

following:

Ten minutes of sanitizing showed 2.9-3.1 log10 reduction of E. coli and Salmonella on lettuce and

tomato and 2.5-2.6 log10 reduction of E. coli and Salmonella on radish.

Fifteen minutes of sanitizing showed 3.6-3.8 log10 reduction of E. coli and Salmonella on lettuce and

tomato and 3.0-3.2 log10 reduction of E. coli and Salmonella on radish.

If greater microbial reduction or reduced processing time is desired, future developments could investigate the

effect of changing some aspects of the system design, including:

Quantity of reactors

Locations of reactors with respect to the processing chamber

Quantity and locations of air flow outlet(s) from the processing chamber

Size of the processing chamber

Tumbling speed of the processing chamber

If increased processing capacity is desired with the single MLE volume, there is volume available within the

current prototype envelope to increase capacity by 50-100%. If increased capacity beyond that is desired, a double

MLE system could be developed.


13

In addition, any future developments of NTSAPP technology would include pumps to provide single-pass flow

of lab air through the system, improved materials and sealing methods for ozone containment, and a catalytic

scrubber on the outlet line for ozone removal.

Testing was completed with trained food quality panelists to investigate the impact of NTSAPP processing on

food quality. Testing with lettuce, tomato, and radish showed no significant change in food quality from 10 minutes

of NTSAPP processing.

Acknowledgments

ORBITEC would like to acknowledge NASA JSC for funding contract number NNX11CG08P and

NNX12CA62C for supporting advancement of these technologies.

References 1Perchonok, M., “Advanced Food Technology Workshop Report Volume I”, NASA Space and Life Sciences Directorate –

Habitability and Environmental Factors Division, 2003, JSC – 29993. 2Buck, J.W., Walcott, R.R., Beuchat, L.R., “Recent Trends in Microbiological Safety of Fruits and Vegetables,” Plant Health

Progress, Online, 2003. 3Berg, G., Eberl, L., and Hartmann, A., “The Rhizosphere as a Reservoir for Opportunistic Human Pathogenic Bacteria,”

Environmental Microbiology, Vol. 7, Issue 11, 2005, pp. 1673-1685 4Morales, A., Garland, J.L., and Lim, D.V., “Survival of Potentially Pathogenic Human-Associated Bacteria in the

Rhizosphere of Hydroponically Grown Wheat,” FEMS Microbiology Ecology, Vol. 20, 1996, pp. 155-162. 5Chang, J., “Recent Development of Plasma Pollution Control Technology: A Critical Review” Science and Technology of

Advanced Materials, Vol. 2, 2001, pp. 571-576. 6Oda, T., “Non-Thermal Plasma Processing for Environmental Protection: Decomposition of Dilute VOCs in Air,” Journal of

Electrostatics, Vol. 57, 2003, pp. 293-311. 7Montie, T.C., Kelly-Wintenberg, K., and Roth, J.R., “An Overview of Research Using the One Atmosphere Uniform Glow

Discharge Plasma (OAUGDP) for Sterilization of Surfaces and Materials,” IEEE Transaction on Plasma Science, Vol. 20, No. 1,

2000, pp. 41-50. 8Gaunt, L.F., Beggs, C.B., and Georghiou, G.E., “Bactericidal Action of the Reactive Species Produced by Gas-Discharge

Nonthermal Plasma at Atmospheric Pressure: A Review,” IEEE Transaction on Plasma Science, Vol. 34, No. 4, 2006, pp. 1257-

1269. 9Laroussi, M., “Mechanisms of Interaction of Cold Plasma with Bacteria,” Fifth International Conference on the Physics of

Dusty Plasmas, American Institute of Physics, 2008. 10Spilimbergo, S., Dehghani, F., Bertucco, A., and Foster, N., “Inactivation of Bacteria and Spores by Pulsed Electric Field

and High Pressure CO2 at Low Temperature,” Biotechnology and Bioengineering, Vol. 82, Issue 1, 2003, pp. 118-125. 11Russel, N.J., Colley, M., Simpson, R.K., Trivett, A.J., and Evans, R.I., “Mechanism of Action of Pulsed High Electric Field

(PHEF) on the Membrane of Food-Poisoning Bacteria is an ‘All-or-Nothing’ Effect,” International Journal of Food

Microbiology, Vol. 55, 2000, pp. 133-136. 12Beuchat, L.R., “Ecological Factors Influencing Survival and Growth of Human Pathogens on Raw Fruits and Vegetables,”

Microbes and Infection, Vol. 4, 2002, pp. 413-423. 13Hummerick, M.E., et. al. “A Hazard Analysis Critical Control Point Plan Applied to the Lada Vegetable Production Units

(VPU) to Ensure the Safety of Space Grown Vegetables,” International Conference on Environmental Systems, AIAA, 2011 14Polowsky, P., and Jimenez-Maroto, L.A., “Vegetable Descriptive Evaluation,” University of Wisconsin Sensory Analysis

Laboratory, Sensory Evaluation Report and Statistics, 2015.

non-thermal fresh food sanitation by atmospheric pressure

Documents