Inactivation of BSL-2 and BSL-3 human pathogens using FATHHOME’s Trinion Disinfector: A rapid and eco-friendly ozone-based dry disinfection approach

Highlights

• •Scientific questions:

• This study examines whether ozone-based dry disinfection with the FATHHOME Trinion Disinfector provides a quick, safe, and effective way to decontaminate protective equipment (PPE) contaminated with biosafety level (BSL)-2 and BSL-3 pathogens. It fills important gaps in current decontamination methods by assessing multi-pathogen inactivation.

• Evidence before this study:

• •Previous studies have shown ozone to be a powerful, broad-spectrum disinfectant, but its use has been limited by lengthy exposure times and limited data on real-world PPE applications. No previous research has thoroughly assessed its effectiveness against both enveloped and non-enveloped viruses, bacteria, and yeast on PPE surfaces under dry conditions.
• •New findings:

• The FATHHOME Trinion Disinfector achieved over 99.9 % inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and adeno-associated virus (AAV), and a 99 % reduction of herpes simplex virus type 1 (HSV-1) and hepatitis B virus (HBV) within 10 min at 60 ppm ozone. Bacterial and fungal pathogens, including Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae, showed more than 90 % reduction after 30 min.

• Significance of the study:

• This work supports ozone as a quick, residue-free, and scalable disinfection method that enables safe PPE reuse, reduces biomedical waste, and offers an eco-friendly alternative to chemical disinfectants. It enhances infection control, lowers costs, and supports sustainability.

Abstract

The role of personal protective equipment (PPE) in protecting against exposure to infectious agents and toxic chemicals is well-established. However, the global surge in PPE demand during the pandemic exposed challenges, including shortages and environmental impacts from disposable waste. Developing effective, scalable, and sustainable decontamination methods for the reuse of PPE is essential. Ozone has emerged as a promising, eco-friendly disinfectant due to its strong oxidative properties, rapid action, and residue-free breakdown into oxygen. This study evaluates the effectiveness of the FATHHOME Trinion Disinfector, an innovative ozone-based dry sterilization device, for inactivating pathogens on PPE materials, such as not resistant to oil 95 (N95) masks and face shields. The device’s bactericidal performance was tested against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Enterococcus durans, Enterococcus faecalis, and Saccharomyces cerevisiae, achieving a 1- to 2-log reduction in these bacterial and fungal pathogens. A 30-minute ozone exposure cycle was found to attain maximum sterilization efficiency. We also demonstrated the disinfector’s efficacy against viral pathogens, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), adeno-associated virus (AAV), herpes simplex virus type 1 (HSV-1), and hepatitis B virus (HBV) on PPE surfaces. SARS-CoV-2 contamination on face shields and N95 masks decreased by 99.9 %, and AAV infectivity was nearly eliminated. Similar reductions were observed for HSV-1 and HBV. Overall, the findings confirm that ozone-based disinfection offers a rapid, scalable, and sustainable method for decontaminating PPE. These results support the establishment of standardized ozone disinfection protocols to enhance infection control, address PPE shortages, and minimize environmental waste.

1. Introduction

The coronavirus disease 2019 (COVID-19) pandemic emphasized the critical role of personal protective equipment (PPE) in protecting healthcare workers and the public from highly transmissible pathogens, including biosafety level 2 (BSL-2) and BSL-3 agents severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 disease, herpes simplex virus (HSV), a common pathogen responsible for recurrent infections, hepatitis B virus (HBV), a major global health threat because its ability to cause chronic liver disease, and adeno-associated virus (AAV), a widely used vector in gene therapy that requires careful handling to prevent unintended exposure [[1], [2], [3], [4], [5]]. Additionally, PPE remains essential in preventing the spread of bacterial pathogens like Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), Salmonella typhimurium (S. typhimurium), Enterococcus durans (E. durans), Enterococcus faecalis (E. faecalis), and the fungal species Saccharomyces cerevisiae (S. cerevisiae) [6]. However, the massive demand for PPE during the COVID-19 pandemic highlighted persistent challenges, such as shortages due to supply chain limitations and the environmental burden of single-use PPE waste [7]. Reusable PPE offers a sustainable alternative; however, its safe reuse requires effective decontamination methods that eliminate pathogens without compromising material integrity and protective function [8]. Traditional decontamination approaches, such as chemical disinfectants (e.g., ethanol, hydrogen peroxide), ultraviolet (UV) irradiation, and autoclaving, face challenges including toxicity, material degradation, incomplete pathogen inactivation, and operational complexity [9,10].

Ozone, a triatomic oxygen molecule with strong oxidative properties, has gained recognition as an effective, fast-acting, and residue-free disinfection [11]. Ozone's broad-spectrum antimicrobial activity against enveloped viruses like SARS-CoV-2, non-enveloped viruses like AAV, Gram-positive and Gram-negative bacteria, and fungi arises from its strong ability to oxidize lipids, proteins, and nucleic acids [[12], [13], [14]]. Its penetration capabilities enable the inactivation of resilient bacterial biofilms on surfaces [13]. Ozone decomposes spontaneously into oxygen, eliminating toxic residues and aligning with eco-friendly disinfection goals [15].

A review article on the potential of ozone to combat SARS-CoV-2 emphasized that ozone disinfection efficacy is highly dependent on both dose and humidity, underscoring the need for research to understand disinfection under variable real-world conditions [16]. Furthermore, while promising results have been reported on ozone’s ability to inactivate SARS-CoV-2 from hard-to-clean office surfaces, these approaches often rely on prolonged exposure, elevated humidity, or lack consistency across surface types [17].

Despite these advantages, a key research gap exists, i.e., the lack of validated, dry ozone disinfection systems that function under ambient conditions and are tested against a comprehensive panel of pathogens, including non-enveloped viruses and fungi, on clinically relevant PPE materials. This gap becomes particularly significant for practical implementation in fast-paced clinical environments, where humidity control is often impractical and rapid turnover is essential [18]. Therefore, while ozone has clear potential, more translational studies are required to assess its applicability in real-world, low-humidity, short-cycle scenarios [18].

FATHHOME’s Trinion Disinfector addresses these gaps by utilizing a novel disinfection device for rapid and efficient pathogen inactivation. This device utilizes controlled, high-concentration ozone in a moisture-free environment, minimizing exposure time while preserving the structural integrity of PPE materials such as not resistant to oil 95 (N95) respirators, gowns, and face shields. A previous study from our group on ozone-based PPE decontamination using FATHHOME's device, with different versions, reported more than 99 % inactivation of human coronavirus OC43 (HCoV-OC43), a close genetic relative of SARS-CoV-2, on N95 masks and face shields at a concentration of 25 parts per million (ppm) for 15 min [[19], [20], [21]]. This study evaluates the FATHHOME's Trinion Disinfector’s performance in inactivating SARS-CoV-2, HSV, HBV, AAV, and bacterial/fungal pathogens (E. coli, P. aeruginosa, S. aureus, S. typhimurium, E. durans, E. faecalis, and S. cerevisiae) on PPE surfaces, including N95 masks and face shields.

The primary goals of this study were to evaluate the effectiveness of the FATHHOME’s Trinion Disinfector on a broad spectrum of microbes, assess material compatibility, and measure the operational efficiency of dry ozone treatment using this device. Our findings aim to provide evidence-based insights into the feasibility of this approach as a rapid, environmentally friendly, and scalable method for disinfection. This study seeks to develop guidelines for ozone-based disinfection protocols, enhance infection control measures, and mitigate the risks associated with pathogenic microorganisms in healthcare settings.

2. Materials and methods

2.1. Bacterial and yeast strains (BSL-2 microorganisms) and their culture conditions

The bacterial and yeast strains used in this study are listed in Table 1. These microorganisms were stored at −80 °C and revived by streaking on appropriate media, including lysogeny broth (LB), yeast peptone dextrose (YPD), or brain heart infusion (BHI) agarose plates (Teknova; Hollister, California), and incubated overnight at the listed temperatures. To evaluate the sensitivity of FATHHOME’s Ozone Dry Disinfector, a single colony from a culture plate was used to inoculate 5 mL of broth in a 15 mL culture tube. The tube was then incubated overnight at 200 revolutions per minute (rpm) at a specific temperature. LB and YPD broth were prepared as described by the manufacturer (BD Difco™; Franklin Lakes, New Jersey), and BHI medium was prepared according to the manufacturer’s guidelines (Teknova; Hollister, California). The following day, the optical density (OD) 600 of the liquid culture was measured at 600 nm using a SPECTRONICTM 200 spectrophotometer (Thermo Scientific^TM; Waltham, Massachusetts), and the culture was diluted to a specific OD 600 nm consistent with culture density between 1 × 10⁶ and 1 × 10⁷. This standardized pathogen stock was utilized in all experiments to evaluate the efficacy of the FATHHOME Trinion Disinfector device. All experiments were performed in triplicate.

Table 1.

List of microbes and their culture conditions.

Organism	Strain	Growth	OD 600	Source
Escherichia coli	MG1655	LB media 37 °C	0.05	Arkin Lab, LBNL
Pseudomonas aeruginosa	mPAO1	LB media 37 °C	0.05	Fleiszig Lab, UC Berkeley
Staphylococcus aureus	ATCC 23,235	LB media 37 °C	0.01	ATCC 23,235
Salmonella typhimurium	ms205	LB media 37 °C	0.05	Celniker Lab, LBNL
Enterococcus durans	BDGP3	BHI media 30 °C	0.01	Celniker Lab, LBNL
Enterococcus faecalis	MIC0000106	BHI media 37 °C	0.05	Celniker Lab, LBNL ATCC 19,433
Saccharomyces cerevisiae	BY4742	YPD media 30 °C	1.00	JBEI-19580

Abbreviations: LB, lysogeny broth; YPD, yeast peptone dextrose; BHI, brain heart infusion; OD, optical density; LBNL, Lawrence Berkeley National Laboratory; Lab, laboratory.

2.2. Viruses and their (BSL-2/3) culture conditions

2.2.1. SARS-CoV-2

Severe acute respiratory syndrome (SARS)-related coronavirus 2, isolate USA-WA1/2020, was obtained through BEI Resources, NIAID, NIH: SARS-related coronavirus 2, isolate USA-WA1/2020, NR-52281. P1 virus stock was prepared by inoculating a monolayer of Calu-3 human lung adenocarcinoma cells (ATCC, cat. # HTB-55) cultured in Dulbecco's modified eagle medium (DMEM) with 10 % fetal bovine serum (FBS) and Pen/Strep in the biocontainment safety level 3 laboratory [22]. Four days later, the supernatant containing virions was collected and centrifuged at 5000 rpm for 5 min at 4 °C to remove any cell debris before being used for the inactivation assay. The number of infectious viruses was assayed by the standard plaque assay for determining the plaque-forming units 9PFU) [23,24].

2.2.2. AAV

This study utilized green fluorescent protein (GFP)-expressing AAV (AAV-GFP) (BEI Resources, NR-52390, adenovirus serotype 5, Clone Ad5-CMV-hACE2/RSV-eGFP). The virus was propagated on a monolayer of the Human embryonic kidney cell line (HEK-293). Approximately 500,000 HEK-293 cells were plated in a 6-well flat-bottom cell culture plate using DMEM with L-glutamine and sodium pyruvate. After a minimum attachment period of 3 h at 37 °C and 5 % CO₂, the cells were infected with the AAV virus, followed by a 1.5-hour incubation at 37 °C and 5 % CO₂. Any unattached viruses were removed from the supernatant, and fresh DMEM medium was added to the cells, which were then incubated at 37 °C and 5 % CO₂. After a 48-hour incubation, the supernatant containing AAV was harvested and centrifuged at 5,000 rpm for 5 min at 4 °C to remove cell debris. The supernatant was then stored at −80 °C until further experimentation. The expression of GFP following the infection of the target cells was used to visualize residual infectious viruses after inactivation by FATHHOME's device.

2.2.3. Herpes simplex virus type 1 (HSV-1)

Human HSV-1 (ATCC-VR-1789) was propagated in vero cells (ATCC; CRL-1586) cultured in DMEM) with L-glutamine and sodium pyruvate (Corning Inc., Corning, New York), 100 U/mL penicillin (Thermo Scientific^TM; Waltham, Massachusetts), and supplemented with 10 % FBS (Sigma-Aldrich, St. Louis, Missouri). Cells were plated for 24 to 48 h to achieve 80 %-90 % confluency before being infected with the culture supernatant containing the HSV-1 virus. The medium was removed, and 100 µL of virus stock diluted in DMEM was inoculated to provide an optimal multiplicity of infection (MOI). The virus was allowed to attach for 1 to 2 h at 37 °C in a humidified atmosphere containing 5 % CO₂. The unattached virus was removed by washing with phosphate-buffered saline (PBS) (Corning, Inc., Corning, New York), and the cell monolayer was then added to fresh DMEM. Cells were incubated for 4 to 5 days at 37 °C and 5 % CO₂, and the culture supernatant was collected by centrifugation to remove cell debris. It was then stored at −80 °C for use in inactivation assays. The amount of infectious HSV-1 was determined by plaque assay on Vero-ACE2-TMPRSS2 cells (ATCC; NR-54970).

2.2.4. HBV

HepG2 cells were maintained in DMEM with L-glutamine and sodium pyruvate (Corning, Inc., Corning, New York) and 100 U/mL penicillin (Thermo Scientific^TM; Waltham, Massachusetts), supplemented with 10 % FBS (Sigma-Aldrich, St. Louis, Missouri). The HepG2 cell line, which produces hepatitis B, genotype A1 (HepG2-GtA1), was obtained from BEI/ATCC. High-titer HBV was prepared from these cells by growing them in DMEM (Corning Inc., Corning, New York) with 100 U/mL penicillin (Thermo Scientific^TM; Waltham, Massachusetts) supplemented with 10 % fetal bovine serum (Sigma-Aldrich, St. Louis, Missouri). Cells were grown to approximately 90 % confluence, and the supernatant was collected to quantify the virus using the GeneJET viral deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) purification kit (Thermo Scientific^TM; Waltham, Massachusetts). The quantification of the HBV genome in the supernatant was performed using the TaqMan microbe detection assay (Thermo Scientific; Waltham, Massachusetts). Inoculation of HepG2 cells was performed to infect them with the specified volume of supernatant containing the HBV infectious virus. The localization of HBV core antigen was determined by an immunofluorescence assay with a mouse anti-core antibody (Thermo Scientific^TM; Waltham, Massachusetts).

2.3. Dry ozone generation for disinfection

This study employed the FATHHOME’s Trinion Disinfector for its ability to create a dry ozone environment for microbial disinfection. The device features a vacuum-based dry disinfection chamber equipped with a computer-controlled ozone generator and a catalytic converter using manganese dioxide and copper oxide (MnO₂–CuO₂). This self-contained system regulates high concentrations of ozone gas in a negative-pressure atmosphere to achieve biocidal effects. The device maintains an ozone gas concentration of 60 ppm, while the vacuum ensures that internal pressure remains between 30 kPa and 15 kPa, preventing ozone from escaping during the sanitization cycle. Once the vacuum seal is established, ozone is generated through the electric cleavage of atmospheric oxygen (O₂) into elemental oxygen (O⁻), which combines with uncleaved oxygen molecules to form ozone. During the experiment, the chamber contents were kept under negative pressure, with the only exit for all gases in the system routed through the MnO₂–CuO₂–ozone scrubber, ensuring environmentally safe exhaust in compliance with the Occupational Safety and Health Administration (OSHA) and Food and Drug Administration (FDA) guidelines. Unlike conventional ozone chambers, which often rely on humidification or require extended exposure times, the FATHHOME’s Trinion Disinfector operates under dry conditions, achieving rapid decontamination cycles. Its compact design, integrated ozone destruction unit, and programmable control system distinguish it from larger, less targeted ozone devices used in industrial settings. For this study, the FATHHOME device was housed in the appropriate BSL containment and controlled remotely from outside the test room for each disinfection cycle. Previous studies evaluated an earlier version of the device for its virucidal and bactericidal efficacy [19,20].

2.4. Experimental setup

2.4.1. Inactivation of bacteria and yeast

Sterilization of commonly used PPE in hospital settings, including N95 respirator masks (DemeTECH G 301-22; Miami Lakes, Florida) and plastic face shields (Sellstrom S 35000; Elgin, Illinois), was tested in this assay. Glass microscope slides (Radnor, Pennsylvania), representing a smooth surface, were used wherever mentioned. N95 respirator masks were cut into 0.5 cm × 0.5 cm square pieces that fit within the wells of a 96- well, flat- bottomed plate (Thermo Scientific Nunclon ™ Delta Surface 168055; Roskilde, Denmark), and face shields were cut into pieces comparable in size to the glass slides (1 inch × 2 inch). Five μL of liquid (representing a cough or respiratory droplet) stock of each pathogen was pipetted onto the corresponding surface, mask piece, face shield piece, or glass slide. Samples were dried in a 37 °C incubator until the liquid culture was thoroughly dried (approximately 1 h). Once dry, experimental plates containing the samples were exposed to ozone gas inside the FATHHOME device for durations and concentrations as specified. Control plates underwent a vacuum cycle of the same length in the FATHHOME device without ozone production. An in-built internal ozone sensor was used to record the ozone concentrations. After exposure to ozone or control conditions, dried cell droplets were resuspended in 100 μL of the appropriate medium (Table 1) and thoroughly mixed using a pipette. Subsequently, a ten-fold dilution was prepared, and 80 μL of each diluted sample was inoculated onto the indicated plate with the referenced media composition. Samples were incubated overnight at temperatures conducive to optimal growth, except for S. cerevisiae, which required an additional 24-hour incubation period to yield observable colonies. The colonies were manually tabulated as colony-forming units (CFUs). The percentage of mortality was calculated by dividing the difference between the control and test counterparts by the number of controls’ CFU. To ensure replicability, 5–7 trials were conducted for each organism on an N95 mask and face shield, and 3–5 trials were conducted on glass slides.

2.4.2. SARS-CoV-2 inactivation assay

A viral stock of SARS-CoV-2 (USA/WA1/2020) was cultivated in DMEM culture medium, following BSL-3 containment protocols and procedures. The 10 μL drops of SARS-CoV-2 viruses were applied to 7 mm diameter pre-cut pieces of N95 face mask material (3 M Company, St. Paul, Minnesota, Cat. # 8210 V) and face shields (Thermo Scientific^TM; Waltham, Massachusetts, Cat. # 494400). The pieces were prepared using a standard hole punch and placed at the centre of wells in a 24-well plate. The plates were then positioned inside the FATHHOME ozone treatment device. Ozone concentration was continuously monitored, and total ozone dosage (ppm×min) was calculated as a function of exposure time and concentration. After ozone exposure for the specified duration, the material was transferred to a 1.5 mL sterile screw-cap tube (MIDSCI, St. Louis, Missouri, Cat. # PR-SC15AC1G) to recover the residual virus for infectivity testing. A 0.5 mL aliquot of sterile medium was added to each tube. Materials inoculated with the same viral load but without ozone treatment were processed identically for controls. The recovered viral suspension from the tubes was inoculated onto Vero-TMPRSS2 cells, and a plaque assay was performed to quantify the residual infectious virus post-ozone exposure.

2.4.3. AAV inactivation assay

To evaluate the effectiveness of the FATHHOME device in inactivating AAV-infected PPE samples, a total of 10 μL of the viral stock (Adenovirus serotype 5, Clone Ad5-CMV-hACE2/RSV-eGFP) encoding enhanced green fluorescent protein was applied to the surface of a round (hole-punched) piece of face shield and an N95 face mask material, following BSL-2 containment protocols and procedures. Each piece was placed in the center of a well within a 24-well cell culture plate and then positioned in the center of the FATHHOME device chamber. Depending on whether the sample was designated for ozone exposure or no ozone exposure, the FATHHOME device was set for the 10-minute cycle time. Regardless of the exposure time, the FATHHOME device underwent a subsequent 5-minute no-ozone purge cycle before the sample inside could be retrieved and processed. After the cycle was completed, sterile PBS was added to each well containing PPE samples, and the ozone-treated and untreated supernatants were collected for subsequent infectivity assays. HEK-293L cells were seeded in a 96-well plate at a density of 30,000 cells per well and incubated for 3 h. Supernatants from the ozone-treated and untreated AAV-infected PPE samples were added to the HEK-293L cells in 10-fold dilutions (3 points). Following a 1.5-hour incubation at 37 °C, the unattached virus was removed, and fresh medium was added. The cells were then incubated for an additional 48 h. GFP-fluorescing cells following AAV infection were quantified via fluorescence microscopy (Leica Microsystems, Inc., Deerfield, Illinois). The assay was conducted in triplicate across three independent experiments.

2.4.4. HSV inactivation assay

The experimental procedures outlined for the AAV assay were used to test the inactivation of HSV. However, plaque-forming assays were employed to quantify the efficacy of disinfection against HSV-infected PPE samples under BSL-2 containment protocols. N95 face masks and face shield pieces were placed in the center of a 24-well plate. After inoculating the PPE samples with HSV, the plate was transferred to the FATHHOME disinfector and subjected to either ozone treatment or untreated conditions. Following treatment, samples were retrieved from the plate. The effectiveness of ozone treatment was determined by assaying the residual live virus (plaque-forming units) through plaque assay.

2.4.5. HBV inactivation assay

The experimental procedures outlined above for AAV and HSV were used for the inactivation of HBV, except for the downstream assay to detect residual infectious virus. After ozone treatment, an immunolocalization assay detected the HBV-infected cells in the FATHHOME device. N95 face masks and face shield materials (cut into circular pieces) were placed in the centre of a 24-well plate. Following HBV inoculation of the PPE samples, the plate was transferred to the FATHHOME device and subjected to either ozone or no ozone treatment. HepG2 cells, maintained in DMEM containing L-glutamine and sodium pyruvate (Corning, Inc., Corning, New York), 100 U/mL penicillin (Thermo Scientific^TM; Waltham, Massachusetts), and 10 % fetal bovine serum (Sigma-Aldrich, St. Louis, Missouri), were plated in a 12-well plate for infection with the recovered virus. The recovered virus was added to the HepG2 monolayer. After a 1.5-hour incubation, fresh medium was added, and the cells were incubated for an additional 24 h to permit viral replication. Residual infectious virus was determined by counting the HBV-infected cells, which were detected via immunolocalization of the HBV core antigen using a mouse anti-core antigen antibody (Thermo Scientific^TM; Waltham, Massachusetts, cat. #MA17607) and anti-mouse AlexaFluor 594 (red) antibody. The number of HBV-infected cells in ozone-treated and untreated samples was quantified through the quantification of red signals to estimate inactivation efficiency.

2.5. Statistical analysis

The data were analysed using Prism 8.0 software (GraphPad Inc., San Diego, CA, USA). The results represent the average of three independent experiments, with error bars indicating the standard deviation, which was calculated using the standard formula in Prism. Statistical comparisons between groups were performed using an unpaired two-tailed t-test or two-way ANOVA, as appropriate.

3. Results

3.1. Assessment of bactericidal (BSL-1/2) efficiency of FATHHOME ozone dry disinfectors

The experiment aimed to assess the effectiveness of FATHHOME's ozone-based dry disinfection device on three common hospital materials: N95 masks, plastic face shields, and glass microscope slides. Select microbes, bacteria, and yeast (E. coli and S. cerevisiae) and common human pathogens, including P. aeruginosa, S. aureus, S. typhimurium, E. durans, and E. faecalis (Table 1), were tested for their sensitivity to ozone treatment by the FATHHOME device. The disinfection cycle was evaluated at three durations: 10, 20, and 30 min, generating −750, 1,500, and 2,200 ppm ozone atmospheres (Fig. 1). The results of microbial sensitivity to ozone, as indicated by the CFU/mL revived, are presented and discussed.

Fig. 1.

Ozone and pressure monitoring inside FATHHOME Trinion Disinfector. Cycle characteristics of 10 min (peak ≈ 750 ppm × min) (A) 20 min (≈ 1,500 ppm × min) (B), and 30 min (≈ 2,200 ppm × min) (C) treatments. Measured levels of O₃ (pink line) and no O₃ (black line), pressure (brown line), with the calculated ppm minute dose. Abbreviations: O₃, ozone; ppm, parts per million; min, minutes.

The data showed a 1-log reduction in E. coli CFU/mL across most material and ozone cycle length combinations. This equates to a sterilization efficacy of over 90.00 % for all trials, except for the 10-minute ozone cycle on glass samples, which achieved a sterilization efficacy of 84.00 % (Fig. 2A–C). Also, an exceptional 4-log reduction in CFU/mL for the 20-minute ozone cycle on face shield samples was observed. As such, it is deemed that 20 min is the optimal cycle length for the sterilization of E. coli-contaminated materials.

Fig. 2.

Effects of 10, 20, and 30 min of ozone exposure on viability of BSL-1 and BSL-2 microbes, under FATHHOME Trinion Disinfector. CFU/ mL after +/- O₃ treatment was tabulated for 6 bacteria and a yeast (cultures were revived from a glass slide, a face shield, and a face mask, respectively). Mortality (%) was calculated by comparing O₃-treated and untreated cultures. A–C) E. coli; D–F) P. aeruginosa; G–I) S. aureus; J–L) S. typhimurium; M−O) E. durans; P–R) E. faecalis; S–U) S. cerevisiae. Abbreviations: BSL, biosafety level; CFU, colony forming unit; O₃, ozone; E. coli, Escherichia coli; P. aeruginosa, Pseudomonas aeruginosa; S. aureus, Staphylococcus aureus; S. typhimurium, Salmonella typhimurium; E. durans, Enterococcus durans; E. faecalis, Enterococcus faecalis; S. cerevisiae, Saccharomyces cerevisiae.

For P. aeruginosa, a 2-log reduction in CFU (sterilization efficacy of over 80 %) was observed across all trials, except for the 20-minute ozone cycle on glass samples, which had a sterilization efficacy of 68.72 % (Fig. 2D–F). However, when the exposure to ozone was increased from 20 to 30 min, the sterilization efficacy on glass increased to 87.82 %. Notably, the reduction in CFU/mL was over 1-log (over 90 % reduction) for P. aeruginosa mask samples across all cycle lengths. Given the variability in results for certain materials, a 30-minute cycle is optimal for sterilizing P. aeruginosa on our materials of interest.

For S. aureus, a 1-log reduction in CFU/mL was observed, indicating a sterilization effectiveness of over 80 % in all trials, except for the 10-minute ozone cycle on glass samples, which showed a slightly lower effectiveness of 79.03 % (Fig. 2G–I). The highest sterilization rate was seen in the face shield samples during the 10-minute ozone cycle, achieving a reduction of just over 1 log in CFU/mL (sterilization effectiveness of 90.80 %); however, the 30-minute cycle for S. aureus was the most effective overall in reducing CFU across all materials.

For S. typhimurium, most sample types showed a 1-log reduction in CFU/mL, with some outliers. The 10-minute ozone cycle achieved sterilization rates of 62.38 % for face shield samples and 74.67 % for glass slide samples. In comparison, all materials exposed to a 30-minute ozone cycle showed more than a 1-log reduction in CFU/mL. Glass slide samples treated with ozone for 30 min experienced nearly a 4-log reduction in CFU/mL compared to control samples. (Fig. 2J–L). The best ozone exposure time for sterilizing S. typhimurium is between 20 and 30 min.

Trials on E. durans showed a 0.5- to 1-log reduction in CFU/mL, demonstrating sterilization rates over 75 % across all materials and ozone cycle durations, except for the 10-minute ozone cycle on glass samples, which was approximately 52.00 % (Fig. 2M–O). Additionally, all E. durans trials on mask samples, as well as the 10- and 20-minute trials on face shield samples, achieved sterilization efficacies of over 85.00 %. Overall, the 20-minute ozone cycle is the most effective for sterilizing E. durans across all materials.

For E. faecalis, a 1.5-log reduction in CFU/mL was observed across all trials, demonstrating a sterilization efficacy of over 80.00 %. However, the 10-minute ozone cycle on mask samples showed a slightly lower efficacy of 78.00 % (Fig. 2P–R). Notably, all samples exposed to ozone for the 30-minute cycle achieved over 1-log reductions in CFU (more than 90.00 % sterilization), establishing the optimal duration of ozone exposure for sterilization of E. faecalis at 30 min.

Sterilization results for S. cerevisiae are remarkably similar across all materials and ozone cycle lengths (Fig. 2S–U). The results show CFU/mL reductions of approximately 1.5-log across all trials, consistent with sterilization efficacies exceeding 90 %. The difference between the average sterilization rate for a 10-minute ozone cycle (93.08 %) and the average for a 30-minute ozone cycle (97.18 %) is 4.10 %. Therefore, the 30-minute ozone cycle is optimal for sterilizing S. cerevisiae.

The results show that the FATHHOME Trinion Disinfector, an ozone-based dry disinfection device, is more than 90.00 % effective at sterilizing many common hospital microorganisms and pathogens on frequently used hospital materials. The system achieves a 99.99 % sterilization rate of microbes by optimizing time and ozone levels for samples across various materials (Fig. 2). Samples on glass slides had the lowest overall sterilization rates. In contrast, most hospital PPE, such as masks or face shields, which require dry sterilization methods, showed higher sterilization effectiveness. Although sterilization effectiveness varied among organisms and materials, we saw over 1-log reductions for most microbial samples. The 30-minute ozone exposure cycle demonstrated strong sterilization ability on all microorganisms and material samples. Further development of the device to increase ozone concentration in the sterilization chamber within 30 min could improve its sterilization efficiency across different microbes and materials.

3.2. Effectiveness of ozone exposure for disinfecting PPE contaminated with SARS-CoV-2 (USA-WA.1/2020)

We evaluated the efficacy of ozone exposure on inactivating SARS-CoV-2 (USA-WA1/2020). This highly infectious pathogen has resulted in one of the most globally disruptive pandemics in modern history, by using the FATHHOME device. The PPEs, face shields, and N95 face masks contaminated with SARS-CoV-2, as droplets, were used to assay the efficacy of virus inactivation. Control samples received no ozone exposure (No ozone). In contrast, treatment samples were exposed to 60 ppm ozone for 10 min, followed by the determination of residual infectious virus as the plaque-forming units. Fig. 3A and 3B demonstrated significantly reduced plaque-forming units in ozone-treated face shields and N95 masks, indicating effective inactivation of SARS-CoV-2 on these surfaces. Quantifying these plaques obtained from the residual infectious viruses showed a 3-log reduction in infectious viruses in ozone-treated PPE sets compared to untreated control sets (Fig. 3C). The results highlight that ozone treatment in the FATHHOME device can significantly reduce the SARS-CoV-2 USA-WA1/2020 virus from the face shields and N95 masks and supports the potential for safer reuse in high-risk settings. The observed correlation between ozone exposure and plaque reduction emphasizes the method’s reliability for decontamination under standardized protocols.

Fig. 3.

Effect of 60 ppm-10 min ozone exposure on SARS-CoV-2 USA- WA1/2020. A–B) Infectious virus particles in No O₃ (control) and O₃ (treated) face shield and N95 face mask supernatants were determined via plaque assay. Supernatant volumes (20 µL, 2 µL, 0.2 µL, and 0.02 µL) derived from USA-WA1/2020 control and test with ozone were tested. C) Quantification of the number of infectious virus particles on face shields and N95 masks after exposure to ozone or no ozone treatment. ^***, P < 0.001. Abbreviations: SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; ppm, parts per million; O₃, ozone; N95, not resistant to oil 95.

3.3. Effectiveness of ozone exposure for disinfecting PPE contaminated with AAV

Next, we determined the efficacy of ozone exposure for decontaminating AAV exposure from the PPE, face shields, and N95 face masks using the FATHHOME device. As done previously, we used our standard 10-minute cycle with 60 ppm of ozone for this assay. We compared the residual infectious AAV virus levels on the above PPE following exposure to ozone and compared them with the control samples without the ozone exposure. Varying amounts of the recovered samples (20 µL, 2 µL, 0.2 µL, 0.02 µL) from the above experimental conditions were used for infecting the target cells and analysing the infected cells through GFP introduced by the virus following infection (Fig. 4A). The relative levels of green fluorescence due to varying amounts of residual AAV-infected cells following infections were quantified via fluorescence microscopy. Fig. 4B shows relative levels of AAV-driven GFP signals in O₃-treated and untreated PPEs (face shields and N95 masks), with a significantly reduced infectious virus in ozone-treated samples. Quantitative estimation shows about 99.90 % AAV inactivation on these surfaces. These results demonstrate that ozone treatment in the FATHHOME device can significantly inactivate AAV on face shields and N95 masks, supporting its potential for PPE decontamination.

Fig. 4.

Effect of 60 ppm-10 min ozone exposure on AAV. A) AAV infectivity in no O₃ (control) and O₃ (treated) face shield and N95 face mask supernatants was determined. Supernatant volumes (20 µL, 2 µL, 0.2 µL, and 0.02 µL) derived from AAV control and test with ozone exposure were tested; B) Quantification of the number of infectious virus particles on face shields and N95 masks after exposure to ozone or no ozone treatment. ^**, P < 0.01. Abbreviations: GFP, green fluorescent protein; AAV, adeno-associated virus; ppm, parts per million; O₃, ozone; N95, not resistant to oil 95.

3.4. Effectiveness of ozone exposure for disinfecting PPE contaminated with HSV

We also assessed the effectiveness of ozone exposure using the FATHHOME device in inactivating HSV-1 on PPE, specifically face shields and N95 masks. Fig. 5A shows representative plaque assay images comparing residual infectious virus on PPE surfaces after ozone treatment with samples that were not exposed to ozone. PPE exposed to 60 ppm ozone for 10 min had a significantly lower number of plaques than the controls, indicating effective inactivation of HSV-1. The quantification of plaque-forming units (PFUs) in samples recovered from face shields and masks without ozone exposure revealed the presence of infectious viruses that were drastically reduced in identical samples exposed to ozone (Fig. 5B), further supporting the efficacy of ozone in inactivating HSV-1 from surfaces. Overall, this study demonstrates that ozone treatment at 60 ppm for 10 min using the FATHHOME device can effectively inactivate HSV-1 from face shields and N95 masks, suggesting its potential for decontaminating PPE.

Fig. 5.

Effect of 60 ppm-10 min ozone exposure on HSV. A) Infectious virus particles in the no O₃ (control) and O₃ (treated) face shield and N95 face mask supernatants were determined via plaque assay. Supernatant volumes (50 µL, 5 µL, and 0.5 µL) derived from the HSV control and test with ozone were tested. B) Quantification of the number of infectious virus particles on face shields and N95 masks after exposure to ozone or no ozone treatment. *, P < 0.05; ^**, P < 0.01. Abbreviations: HSV, herpes simplex virus; ppm, parts per million; O₃, ozone; N95, not resistant to oil 95.

3.5. Effectiveness of ozone exposure for disinfecting PPE contaminated with the hepatitis B virus

We further evaluated the effect of ozone exposure on the inactivation of HBV present on PPE using our FATHHOME device. Similar to previous methods, the face shield and an N95 face mask contaminated with droplets carrying the infectious virus were subjected to ozone inactivation, followed by recovery of any residual virus to assess the efficacy of HBV inactivation from the PPE surfaces. The amounts of residual infectious viruses were estimated by counting the number of infected cells defined by the presence of a red signal for viral (HBV core) antigen present in the cells. Fig. 6A shows a representative immunofluorescence image detecting HBV core antigen (appearing as red fluorescence in infected cells), with cell nuclei counterstained using 4′,6-diamidino-2-phenylindole (DAPI) (blue fluorescence). The results demonstrated a significant decrease in HBV-positive cells from the samples recovered from PPE exposed to ozone at 60 ppm for 10 min compared to those with no ozone exposure. Quantifying the number of HBV-infected cells from the residual virus in ozone-exposed samples (treated) versus those without ozone exposure (control) indicated a substantial reduction in live HBV, with the most pronounced decrease observed on the face shield. These findings suggest that ozone treatment can effectively reduce HBV contamination from PPE.

Fig. 6.

Ozone treatment reduces HBV contamination on PPE using the FATHHOME device. A) Immunofluorescence analysis of HBV-infected cells (red fluorescence, core antigen) and cell nuclei (blue, DAPI counterstain) under conditions with or without 60 ppm O₃ exposure. Merged panels illustrate reduced HBV-positive cells in O₃-treated samples compared to untreated controls (no O₃). B) Quantification of the number of infectious virus particles on face shields and N95 masks after exposure to O₃ or no O₃ treatment. *, P < 0.05; ^**, P < 0.05. Abbreviations: HSV, herpes simplex virus; ppm, parts per million; O₃, ozone; N95, not resistant to oil 95; HBV, hepatitis B virus; PPE, personal protective equipment; DAPI, 4′,6-diamidino-2-phenylindole. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4. Discussion

The findings of this work established that the FATHHOME Trinion Disinfector is an innovative ozone-based dry disinfection system that can inactivate a range of BSL-2 and BSL-3 human pathogens on common PPE materials. This technology presents a promising alternative to traditional decontamination methods by combining high efficacy and environmental sustainability. Unlike chemical disinfectants such as ethanol and hydrogen peroxide, which produce hazardous waste and may leave toxic residues, ozone is a residue-free, eco-friendly agent [25]. A key advantage of the FATHHOME system is its ability to break down ozone into oxygen after treatment, eliminating the risk of residual toxicity and reducing the need for post-treatment cleaning. These features highlight its potential to reduce both environmental and operational burdens in clinical and laboratory settings.

Our data showed that exposure to ozone can significantly reduce the viability of bacterial pathogens commonly found in healthcare settings. The decrease in CFUs across multiple pathogens indicates that the FATHHOME disinfector offers a high level of sterilization, with an efficacy rate of over 90.00 % in most trials. Notably, a 30-minute exposure cycle consistently achieved the highest reduction rates, especially for E. faecalis and S. cerevisiae, where sterilization rates exceeded 97 %. These findings align with previous studies that emphasize ozone’s strong oxidative ability to disrupt bacterial cell walls, proteins, and nucleic acids, causing irreversible damage and inactivation [25,26]. Ozone's ability to penetrate bacterial biofilms further enhances its potential as a disinfectant, especially in clinical environments where persistent biofilms contribute to resistance to inactivation [[27], [28], [29]].

Since ozone is produced continuously and kept within ± 5 ppm of the setpoint (60–75 ppm) during each cycle, concentration and exposure time are inherently connected in this system. The disinfection effectiveness depends on the total ozone dose delivered, which increases over time (e.g., -750 ppm∙minutes at 10 min, -1,500 ppm∙minutes at 20 min, and -2,200 ppm∙minutes at 30 min). Therefore, improvements in microbial inactivation cannot be solely attributed to exposure time. Future studies that independently vary ozone concentration and exposure duration will be necessary to understand their individual roles in disinfection effectiveness.

The FATHHOME Trinion Disinfector also effectively inactivated viral pathogens, including SARS-CoV-2, AAV, HSV-1, and HBV. Our study demonstrated that a 10-minute exposure to 60 ppm ozone can significantly reduce the levels of infectious viruses on contaminated PPE. We achieved a 3-log reduction (99.90 %) of SARS-CoV-2 and AAV, and a 2-log reduction (99.00 %) for HSV. HBV also showed a substantial decrease in residual infectious viruses, especially on face shields and solid surfaces, highlighting FATHHOME’s potential for decontaminating PPE to reduce the risk of virus transmission. These results confirm that our device can effectively inactivate both enveloped and non-enveloped viruses by oxidizing viral lipid envelopes and capsid proteins, making them non-infectious [30,31]. A 3-log (99.90 %) viral reduction meets the Centers for Disease Control and Prevention (CDC) decontamination standards for general PPE reuse in non-sterile settings. However, for critical-care or sterile applications, combining ozone with additional methods, such as ultraviolet-C (UV-C) treatment or mild heat, could improve pathogen removal and meet stricter decontamination requirements.

Although ozone's antimicrobial effects are well-supported and widely studied, the novelty of this work lies in using a dry ozone-based system under ambient humidity, overcoming the limitations of previous studies that relied on elevated humidity or surrogate viruses. We demonstrate effective decontamination of both enveloped and non-enveloped viruses on real-world PPE materials without leaving chemical residues. The FATHHOME device’s combination of high-concentration ozone, short exposure cycles, and dry operation mode makes it particularly suitable for high-throughput clinical deployment.

Understandably, there are concerns about maintaining the structural integrity of PPE materials after repeated disinfection cycles with ozone. Although we did not specifically evaluate material degradation, previous studies have shown that short ozone exposure cycles at controlled concentrations do not significantly affect the protective function of N95 respirators, face shields, or gowns [32,33]. We plan to investigate the long-term effects of repeated ozone exposure on PPE materials to ensure they remain usable in future studies. Another critical point is the variability in disinfection effectiveness across different materials. We found that glass microscope slides had lower sterilization rates compared to face shields and masks, likely due to differences in ozone diffusion and porosity. This indicates that optimizing ozone concentration and exposure time for different PPE materials should be considered to improve disinfection results.

While the system achieved high efficacy, we acknowledge that 1–2 log reductions seen in bacteria like E. durans may fall short of sterilization benchmarks. However, these reductions are meaningful in moderate-risk settings, especially when part of a multi-layered infection control strategy. Compared to UV-C, hydrogen peroxide vapor, or autoclaving, the FATHHOME system offers shorter cycles, no chemical waste, and better PPE material compatibility. Table 2 contextualizes its performance along with other key disinfection methods, considering factors such as cycle time, residue generation, humidity requirements, PPE material compatibility, and pathogen inactivation level [28,[33], [34], [35], [36]]. This benchmarking highlights both the strengths and limitations of ozone-based disinfection relative to UV-C, hydrogen peroxide vapor, and autoclaving, reinforcing its practical value for rapid and sustainable PPE decontamination.

Table 2.

Comparison of common PPE disinfection methods.

Method	Disinfection time	Residue-free	Humidity needed	PPE compatibility	Cost	Log reduction (virus/bacteria)
Ozone (FATHHOME)	10–30 min	Yes	No	High	Low	3-log / 1–2-log
UV-C	30–60 min	Yes	No	Medium	Low	1–2-log / variable
Hydrogen peroxide vapor	1–2 hrs	No	Yes	Medium	High	4–6-log / 4–6-log
Autoclaving	1 hr	No	Yes	Low (masks fail)	Medium	6-log / 6-log

Abbreviations: PPE, personal protective equipment; UV-C, ultraviolet-C; hr, hour; min, minutes. Log reduction refers to a 10-fold decrease in the number of viable microorganisms after a disinfection or sterilization treatment.

While our study provides strong evidence supporting the efficacy of the FATHHOME Trinion Disinfector for inactivating a wide range of pathogens on PPE surfaces, we acknowledge several limitations. First, we did not assess the structural integrity or filtration performance of PPE materials following repeated ozone exposures. Although previous research has shown that short, controlled ozone treatments (≤30 min) do not significantly degrade N95 respirators or compromise their fit or filtration efficiency [32], further studies are needed to confirm long-term durability across multiple decontamination cycles. Second, our study focused on surface inoculation using controlled droplet volumes, which may not fully replicate the diverse contamination patterns observed in real-world healthcare settings. Third, while our results demonstrate broad-spectrum efficacy, we did not include particularly resistant pathogens such as Mycobacterium tuberculosis or Clostridioides difficile, which possess additional structural defenses and may require higher ozone doses or longer exposures. Prior studies have suggested ozone’s potential against these pathogens [37], but standardized protocols are still lacking. Lastly, the efficacy of our device on multilayered or intricately folded PPE remains to be tested. Future work will aim to evaluate ozone penetration into such complex geometries and explore engineering improvements to optimize ozone flow distribution and concentration uniformity. These efforts will be essential for advancing the practical deployment of dry ozone disinfection systems in diverse healthcare environments.

5. Conclusions

This study demonstrates the effectiveness of the FATHHOME Trinion Disinfector, an ozone-based dry sterilization device, in inactivating a broad spectrum of BSL-2 and BSL-3 pathogens on commonly used PPE, including N95 masks and face shields. The results show that a 10-minute exposure to 60 ppm ozone effectively inactivates viral contaminants such as SARS-CoV-2, HSV-1, HBV, and AAV, achieving a 99.9 % reduction in infectivity. Bacterial and fungal pathogens (E. coli, P. aeruginosa, S. aureus, S. typhimurium, E. durans, E. faecalis, and S. cerevisiae) exhibited over 90.00 % microbial reduction with an optimal 30-minute ozone exposure cycle. It should be noted that although the FATHHOME system effectively reduced viral contamination, the 1- to 2-log reduction observed for some bacterial species may not suffice for applications demanding complete sterilization. This limitation must be considered when defining disinfection standards for different biosafety levels. These findings highlight ozone disinfection as a rapid, scalable, and environmentally sustainable method for PPE decontamination, reducing reliance on chemical disinfectants and mitigating PPE shortages in healthcare and laboratory settings. Future optimizations in ozone concentration and exposure time may further enhance the technology's efficacy for inactivating broad-spectrum pathogens. These results support the development of standardized ozone-based disinfection protocols to strengthen infection control measures and mitigate antimicrobial resistance strategies.