The Portable Chemical Sterilizer (PCS) is a revolutionary, energy-independent, almost waterless sterilization technology for Army medical units. The PCS generates chlorine dioxide from dry reagents mixed with water on-site, at-will, and at point-of-use (PoU) in a plastic suitcase. The Disinfectant–sprayer for Foods and ENvironmentally-friendly Sanitation (D-FENS) and the Disinfectant for ENvironmentally-friendly Decontamination, All-purpose (D-FEND ALL) produce aqueous chlorine dioxide in a collapsible spray bottle and other potential embodiments. These versatile decontamination technologies kill microbes in myriad diverse Dual-use applications for military and civilian consumers.
There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary Service Support Detachments, Combat Support Hospitals, and Area Medical Laboratories to sterilize surgical instruments and to sterilize pathological specimens prior to disposal in operating rooms, emergency treatment areas, and intensive care units. The following ensemble of novel, ‘clean and green’ chlorine dioxide technologies are versatile and flexible to adapt to meet a number of critical military needs for decontamination6,15. Specifically, the Portable Chemical Sterilizer (PCS) was invented to meet urgent battlefield needs and close critical capability gaps for energy-independence, lightweight portability, rapid mobility, and rugged durability in high intensity forward deployments3. As a revolutionary technological breakthrough in surgical sterilization technology, the PCS is a Modern Field Autoclave that relies on on-site, point-of-use, at-will generation of chlorine dioxide instead of steam. Two (2) PCS units sterilize 4 surgical trays in 1 hr, which is the equivalent throughput of one large steam autoclave (nicknamed “Bertha” in deployments because of its cumbersome size, bulky dimensions, and weight). However, the PCS operates using 100% less electricity (0 vs. 9 kW) and 98% less water (10 vs. 640 oz.), significantly reduces weight by 95% (20 vs. 450 lbs, a 4-man lift) and cube by 96% (2.1 vs. 60.2 ft3), and virtually eliminates the difficult challenges in forward deployments of repairs and maintaining reliable operation, lifting and transporting, and electrical power required for steam autoclaves.
The PCS technology proceeds from where no commercial device existed previously and generates the disinfectant chlorine dioxide (ClO2) that has a proven ability to kill vegetative pathogens on fresh produce3,6,9-13,15 or to decontaminate bacterial spores.6,14,15,17 The PCS has been laboratory validated specifically to effectuate sterilization against live cultures of Geobacillus stearothermophilus (GS) spores (see related reference8) and spore bio-indicators of G. stearothermophilus and Bacillus atrophaeus (BA)6,15,16. The PCS has also been adapted to operate with less stringent conditions to ensure Food Safety by inactivating the vegetative pathogens Listeria monocytogenes and Escherichia coli on fresh produce, such as whole tomatoes, and to extend the shelf-life of fresh-cut produce, for example, by inactivating the polyphenoloxidase browning enzyme in sliced apples6,15. To generate chlorine dioxide, the PCS uses novel effector chemistry that proceeds via oxidation-reduction reactions at near-neutral pH, thus eliminating the use of acids and the inherent difficulties of shipping, storing, handling, and disposing acidic wastes in far-forward military deployments1,2,4,17. In addition to the military, the PCS can also be used by Homeland Security/Defense; during natural disasters (Superstorm Sandy, tsunamis, hurricane Katrina) that incapacitate access to power, potable water, and waste removal; on-site by emergency first-responders; and in community hospitals or schools during power outages (blackouts and brown-outs).
The Disinfectant–sprayer for Foods and ENvironmentally-friendly Sanitation (D-FENS) also uses effector chemistry (3 chemical components) and a 2-step mixing process (i. pre-concentration followed by ii. post-reaction dilution) to generate aqueous chlorine dioxide, primarily in a collapsible spray bottle for decontaminating surfaces of Army materiel, food handling equipment and field feeding equipment in Army field kitchens and sanitation centers and Navy Galleys, medical units, showers, and latrines anywhere large numbers of deployed personnel co-exist in close proximity5,6. Validation testing showed that D-FENS eliminates the pathogen Staphylococcus aureus, a common foodborne pathogen, on porous surfaces14. “D-FEND ALL” (Disinfectant for ENvironmentally-friendly Decontamination, All-purpose) provides a simpler (2 chemical components), more convenient (1-step mixing) alternative with unmatched versatility for producing aqueous chlorine dioxide to decontaminate bacterial spores on textiles, for surface disinfection to promote sanitation and hygiene, and to improve water quality and safety, with particular advantages for applications requiring the rapid production of large volumes of dilute chlorine dioxide solutions using small quantities of starting materials for applications in novel graywater recycling technologies designed to generate clean, potable water for Expeditionary Base Camps2.
A variety of mechanisms exist in accordance with the Federal Technology Transfer Act to facilitate the transfer of federal technologies to nonfederal entities as a way to encourage the development and commercialization of technologies for the material benefit of the nation. Accordingly, with their burgeoning potential for many military and civilian uses, the PCS, D-FENS, and D-FEND ALL technologies have been patented and transferred to industry for commercialization via Patent Licensing Agreements and Commercial Evaluation Licenses. A slow, controlled release version of D-FENS (called “D-FENS Lite”) was Technology Transferred to commercial industry for incorporation into packaging materials to extend the shelf-life of fresh berries, and the PCS has also been Technology Transferred to academia and other government agencies for comparative testing with other technologies, for research on Food Safety with fresh produce commodities, and for enhancing undergraduate science education. The Technology Transfer of the PCS and its chemistry led to a commercial product approved for bio-hood sterilization with improvements in time, cost, and environmental protection compared to conventional formaldehyde treatments.
1. The Portable Chemical Sterilizer (PCS)
2. “D-FENS”
3. The PCS for Fresh Fruits and Vegetables
The ability of reduced PCS treatments to kill harmful foodborne pathogens (E. coli and L. monocytogenes) on fresh produce was tested using a spot-inoculation method in which high levels of pathogens were spotted onto the exterior surfaces of tomato wedges.
4. “D-FEND ALL”
The easy-to-operate PCS was designed to achieve sterility by inactivating bacterial spore suspensions or bacterial spore bio-indicators in 30-minute treatments involving the controlled production of chlorine dioxide by unique effector chemistry. Specifically, microbiological validation studies verified that the PCS achieved sterility by inactivating bio-indicators containing spores (105 spores/ml) of either G. stearothermophilus or B. atrophaeus, that are intended to indicate sterilization by steam autoclaves (wet heat) or ethylene oxide gas, respectively. The PCS also sterilized suspensions of G. stearothermophilus spores and B. atrophaeus spores configured as suspensions or dried on hard, non-porous surfaces made of glass or metal and recovered using commercial Difco HY-Check swab kits. G. stearothermophilus spores exposed to the chlorine dioxide treatments were not recoverable but retained their phase bright character after treatment, indicating that the mechanism of spore inactivation by chlorine dioxide is different from wet heat or high pressure and most likely involves damage to the spore’s inner membrane17.
The PCS was adapted for less stringent conditions of ClO2 generation to treat fresh produce to achieve food safety without compromising food quality. Specifically, spot-inoculating high levels of the pathogens E. coli ATCC 11229 (106 CFU/g) and L. monocytogenes OSY-8578 (105 CFU/g) on the exterior surfaces of 25 g samples of tomato wedges, air-drying the samples in a sterile bio-hood for 15 min, placing them inside the PCS, and testing them under various conditions of ClO2 concentration and exposure time (Figure 5) confirmed the efficacy of the PCS for fresh and fresh-cut produce. G. stearothermophilus and B. atrophaeus spore bio-indicators and uninoculated apple slices were placed in the PCS during treatment. Treatment conditions were found that inactivated the target microorganisms on whole tomato surfaces to render safe-to-eat tomatoes free from these foodborne pathogens without compromising the red color of the tomatoes (Table 1). Sliced apples treated in the PCS did not exhibit browning of the pulp tissue when exposed to ambient air subsequently, but remained white for at least a week. The PCS treatment inactivated the polyphenoloxidase enzyme that causes cut apple tissue to turn brown. Also, the apple skin tended not to discolor by exposure to the ClO2.
In addition to inactivating enzymes and vegetative pathogens, ClO2 also inactivates bacterial spores such as Bacillus anthracis (causative agent of ‘Anthrax’) for bio-weapon decontamination. Figure 6A shows the use of Atomic Force Microscopy (AFM) to characterize ClO2-treated B. subtilis spores. The ClO2-treated spores remained intact and did not collapse upon air-drying. These high resolution AFM images also show that the spore coat architecture and topology were unaltered by the ClO2 treatment. For purposes of illustration, Figure 6B shows an electron micrograph of a dormant Bacillus cereus spore with a prominent exosporium layer, which is also characteristic of B. anthracis spores.
Validation experiments showed that the D-FENS sprayer system completely inactivated the infectious bacteria S. aureus, as evidenced by the prevention of growth of black colonies on BPA. In similar tests, 100 ppm chlorine dioxide solutions effected a > 7-log reduction of a 3-strain cocktail of S. aureus inoculated onto stainless steel surfaces after 1, 3, and 5 min of contact. D-FEND ALL provides additional versatility as a convenient method for generating ClO2 with specific advantages for using it in dilute concentrations for applications such as water safety, wastewater disinfection, and textile decontamination. Validation experiments with D-FEND ALL showed the inactivation of bacterial spores without damaging the textile material.
In comparative testing of D-FENS with three commercially available generators which rely on electrochemical cells and selectively permeable membranes to produce different chemical sanitizers (Electrolyzer, WaterStar and Tersano). In these cases, soft water is recommended for use due to the potential for mineral deposits (slaking) that can damage electrochemical cells. Table 2 provides details about each system’s active agent and the advantages of D-FENS in terms of efficacy, power consumption, weight, size, and reduced capital costs compared to other technologies. A concentration of 100 ppm ClO2 was chosen for the D-FENS system (D-FENS can be adjusted to produce ClO2 concentration in the range of 5-4,000 ppm). The D-FENS solution effected a > 7-log reduction of the 3-strain cocktail of S. aureus inoculated onto stainless steel surfaces in contact times as short as 1 min (Table 3). These results demonstrate that D-FENS is an effective disinfectant spray to reduce S. aureus from hard, non-porous contact surfaces in the food preparation and handling environment, such as counter tops, cutting boards, utensils, etc.
Figure 1. The PCS prototype derives from a rigid plastic PELICAN™ suitcase embellished with several design features to ensure sterilization while controlling pressure and temperature, and without off-gassing into the environment15.
Figure 2. The PCS operates via an easy 3-step procedure. Step 2.1: add dry chemical reagents and water in vessel and close suitcase, Step 2.2: let reaction generate ClO2 and treat for 25-60 min, and Step 2.3: air flush through scrubber, then retrieve sterile surgical instruments15.
Figure 3. The D-FENS sprayer (left) generates aqueous ClO2 on-site and at point-of-use in a spray-bottle and easily sprays ClO2 onto contact surfaces (right) to wipe away contaminating pathogens in Army Field Kitchens (center top and bottom)15.
Figure 4. Visible growth of S. aureus colonies on untreated agar surfaces (left), and “no-growth” on agar surfaces treated with the D-FENS ClO2 sprayer.
Figure 5. A typical PCS set-up to inactivate E. coli or L. monocytogenes on exterior tomato surfaces (center), browning enzymes in apple slices (right), and spore bio-indicators (back left and back right)15.
Figure 6. A) AFM of ClO2-treated B. subtilis spores. B) Transmission EM of B. cereus spores with a prominent exosporium14.
ClO2 treatment | Time (min) | GS indicator | BA indicator | Listeriaa (105) | E.colib (106) |
I | 30 | neg. (×3) | neg. (×2) | ||
II | 30 | neg. (×1) | neg. (×1) | neg. (×3) | |
III | 60 | neg. (×1) | neg. (×2) | neg. (×2) |
Table 1. PCS treatments with different ClO2 concentrations (I > II > III) of G. stearothermophilus (GS) and B. atrophaeus (BA) spores and vegetative pathogens (negatives indicate no survivors post-treatment)15.
a. 25g tomato wedges inoculated with Listeria monocytogenes OSY- 8578
b. 25g tomato wedges inoculated with Escherichia coli ATCC 11229
Technology | Active agent | Power (W) |
Weight (lbs) |
Dimensions |
ElectroCide (Electrolyzer Corp.) | HOCl | 84 | 40 | 18” × 12.5” × 6.5 |
FC-2 (WaterStar) | H2O2 | 120 | 6.7 | 12” × 9” × 15” |
Lotus (Tersano) | O3 | 60 | 21 | 12” × 7” × 12” |
SAL-40 (MIOX) | Mixed oxidants | 84 | 150 | 67.3” × 29” × 9.5” |
“D-FENS” | ClO2 | 0 | < 1 | 1 Liter pouch |
Table 2. Sanitizing Solution Generators.
Before Treatment | After Treatment | ||||
Disinfectant | ppm | pH | CFU before | CFU after | log reduction |
S. aureus | |||||
HOCl | 88.0 | 3.0 | 3.4 × 107 | 0.0 | 7.5 |
Clorox | 200.0 | 10.1 | 3.4 × 107 | 0.0 | 7.5 |
H2O2 | 3.4 × 107 | 3.2 × 103 | 3.4 | ||
O3 | 1.8 × 108 | 3.2 × 103 | 4.8 | ||
ClO2 | 100 | 6.7 | 0.0 | > 7 |
Table 3. Efficacy of sanitizers on cells adhered to stainless steel surface.
* This value holds for contact times of 1, 3, and 5 min. Data provided courtesy of Edmund Powers, US Army – NSRDEC.
This foundational R&D has set new research and technical directions through collaborations with academia, other Government agencies, and industry that have led to the commercialization of novel, environmentally-friendly (“green”) technologies. Chlorine dioxide is the first method approved by the National Sanitation Foundation in 20 years for safer, faster, and more environmentally-friendly sterilization than conventional treatments. The PCS, D-FENS, and D-FEND ALL prototypes have been validated as bench-scale prototypes in a laboratory environment, and the range of results imparts these systems with tremendous breadth of versatility to flexibly accommodate the needs of individual users. At this stage, prototypes have been patented and licensed to commercial industry for production and commercialization – commercial products based on these technologies are currently available in the marketplace. Industry partners are needed to make these technologies a reality for the military (and for Homeland Defense/Security, Global Disaster Relief, and Third-World Aid – “Doctor’s Without Borders”), first by determining the application, configuration, and packaging as a salable mixed-chemical technology. For the PCS, FDA clearance testing will be required as a medical sterilization device, but should be attainable, and the resultant device would be procurable by military agencies and have benefits for medical/surgical devices made of plastic, with bendable optics, or with small lumens that are otherwise susceptible to damage by conventional wet heat treatments of “Bertha” autoclaves. D-FENS and D-FEND ALL are convenient spray-and-wipe applications of aqueous chlorine dioxide that eradicate from surfaces vegetative pathogens, viruses, resistant bacterial spores, and bio-films on surfaces to meet important Army needs for decontamination. This corpus of research and development encompasses 15 patents/patent applications, more than seven (7) Technology Transfer agreements, and the 2009 IFT Press/Wiley-Blackwell book “Microbial Safety of Fresh Produce.”
Together, the PCS, D-FENS, and D-FEND ALL provide a versatile and adaptable arsenal of ClO2 technologies for eliminating vegetative pathogens and bacterial spores in myriad applications for surgical instrument sterilization, textile decontamination, fresh and fresh-cut produce sanitization, and hard surface decontamination anywhere microbial contamination is an issue. Thus, invented and validated at the US Army Natick Soldier RDEC laboratories to empower Soldiers with germ-killing strength, to unburden logistics by eliminating the need to transport water, and to protect Soldiers and the environment by decreasing fossil fuel consumption during shipping, reducing CO2 emissions and carbon footprint, and decreasing landfill wastes, these technologies have been commercialized and are available for dual-use applications for civilian consumers.
The authors have nothing to disclose.
The authors would like to acknowledge their gratitude to the U. S. Army Environmental Quality 6.1 Basic Research program, the US Army Institute of Surgical Research, and NSRDEC’s Continuous Product Improvement program and Expeditionary Base Camp TecD for funding this work. We are grateful to Adam Driks (Loyola University Medical Center) for the micrograph shown in Figure 6B.
Sodium chlorite | Sigma-Aldrich | 244155 | |
Sodium sulfite | Sigma-Aldrich | 239312 | |
Sodium ascorbate | Sigma-Aldrich | A7631 | |
Potassium phosphate | Sigma-Aldrich | P0662 | |
Dextrose | Fisher Scientific | D-16 | |
BT Sure biological indicator (steam) | Thermo Fisher Sci | AY759X3 | |
EZ Test (EtO) | SGM Biotech Inc | EZG/6 | |
Difco Hy-check | Becton-Dickinson/ Difco | 290002 | |
Tryptic Soy Agar | Difco | 236950 | |
Nutrient Agar | Difco | 213000 | |
Baird-Parker Agar | Difco | 276840 | |
Egg Yolk-Tellurite | Difco | 277910 | |
0.5% Yeast extract | Difco | 212750 | |
Bacto-Peptone | Difco | 211677 | |
Bacto-Tryptone | Difco | 211705 | |
Agar | Difco | 214010 | |
Soluble starch | Difco | 0178-17 | |
Lab Lemco Beef Extract | Oxoid | L29 | |
Masticator – Classic | IUL Instruments | Cat. No. 400 | |
Stomacher bags | Seward | Stomacher ‘400’ bags |