key: cord-0967128-43o3zm41
authors: Żywicka, Anna; Ciecholewska-Juśko, Daria; Szymańska, Magdalena; Drozd, Radosław; Sobolewski, Peter; Junka, Adam; Gorgieva, Selestina; El Fray, Miroslawa; Fijałkowski, Karol
title: Argon plasma-modified bacterial cellulose filters for protection against respiratory pathogens
date: 2022-04-29
journal: bioRxiv
DOI: 10.1101/2022.04.28.489859
sha: 36bb0bb0793f8737149686dc813cc42bfc5673a7
doc_id: 967128
cord_uid: 43o3zm41

Due to the global spread of the SARS-CoV-2 virus and the resultant pandemic, there has been a major surge in the demand for surgical masks, respirators, and other air filtration devices. Unfortunately, the fact that these filters are made of petrochemical-derived, non-biodegradable polymers means that the surge in production has also led to a surge in plastic waste. In this work, we present novel, sustainable filters based on bacterial cellulose (BC) functionalized with low-pressure argon plasma (LPP-Ar). The “green” production process involved BC biosynthesis by Komagataeibacter xylinus, followed by simple purification, homogenization, lyophilization, and finally LPP-Ar treatment. The obtained LPP-Ar-functionalized BC-based material (LPP-Ar-BC-bM) showed excellent antimicrobial and antiviral properties, with no cytotoxicity versus murine fibroblasts in vitro. Further, filters consisting of three layers of LPP-Ar-BC-bM had >99% bacterial and viral filtration efficiency, while maintaining sufficiently low airflow resistance (6 mbar at an airflow of 95 L/min). Finally, as a proof-of-concept, we were able to prepare 80 masks with LPP-Ar-BC-bM filter and ~85% of volunteer medical staff assessed them as good or very good in terms of comfort. We conclude that our novel sustainable, biobased, biodegradable filters are suitable for respiratory personal protective equipment (PPE), such as surgical masks and respirators. Further, with scale-up, they may be adapted for indoor air handling filtration in hospitals or schools. Graphical abstract

Due to the global spread of the SARS-CoV-2 virus and the resultant pandemic, there has been a major surge in the demand for surgical masks, respirators, and other air filtration devices.

Not surprisingly, this has led to a significant increase in their industrial production 1 .

Importantly, the SARS-CoV-2 pandemic has also led to a renewed recognition of the danger posed by airborne transmission of respiratory pathogens 2 , meaning that the demand for air filtration materials, whether for personal protective equipment (PPE) or indoor air handling will remain high. In this context, the primary focus is on particle filters that trap solid and liquid aerosols, particularly those containing biological contaminants (fungi, bacteria, viruses), although filters that protect against both gaseous and particulate contaminants are also possible 3 . Depending on the type of filter and the properties of the material used, the level of particle filtration efficiency (PFE), bacterial filtration efficiency (BFE), or viral filtration efficiency (BFE) may differ significantly 4, 5 . Currently, the majority of filters use fibers composed of polypropylene (PP), poly(ethylene terephthalate)(PET), poly(tetrafluoroethylene), (PTFE), polyamide (PA), or polycarbonate (PC) 6 . As a result, the surge in production of air filters sparked by the SARS-CoV-2 pandemic has a large carbon footprint, while also generating a surge in non-degradable plastic waste 1,7 -all while the world is already grappling with the environmental impact of millions of tons of existing nondegradable plastic waste 8 .

Therefore, there is a clear and urgent need to develop novel filter materials with sustainability in mind, with the ultimate goal of facilitating a circular economy in this product area. Ideally, such filters would consist of biodegradable materials that can be obtained sustainably, from renewable resources. One example of more sustainable filter material is plant cellulose. However, to increase filtration efficiency, plant cellulose is frequently modified using synthetic polymers e.g. poly(ethyleneimine) and cellulose derivatives, such as cellulose nitrate, mixtures of cellulose esters, cellulose triacetate, or cellulose acetate phthalate 6, [9] [10] [11] [12] .

Additionally, while it is renewable and biodegradable, the overall life cycle of plant cellulose has a high global warming impact plus added drawbacks, including pesticide use and high arable land and water requirements 13 . Biotechnology offers a promising alternative: bacterial cellulose (BC), a biopolymer produced by a spectrum of aerobic, non-pathogenic, Gramnegative bacteria, such as Komagataeibacter xylinus. Importantly, BC consists of loosely arranged, highly crystalline fibrils ~100 nm in diameter and is characterized by high mechanical strength, chemical stability, biocompatibility, and biodegradability [14] [15] [16] . Combined these aspects make BC well suited as an alternative to synthetic polymers used as filtration membranes.

Filtration efficiency is the most important parameter in terms of air filter usability and helps determine potential applications. However, it is important to note that pathogens that are just trapped physically within a filter can still pose a biological hazard, because of the risk that they could be released back into the airstream. As a result, many studies have been devoted to fabricating air filters that not only sequester pathogens but also kill them. For example, filters have been proposed that incorporate antimicrobial agents such as ε-polylysine and natamycin, silver and zinc nanoparticles, or plant extracts from eucalyptus, grapefruit, or propolis 15, [17] [18] [19] [20] .

Likewise, several patents describe BC-based air filters that include antimicrobial components, such as silver 21 , zeolite-supported silver nanoparticles 22 , or chitosan 23 . However, the emergence and spread of microbes resistant to chemical agents 24 , combined with the need to improve sustainability, motivate further research and development into greener BC surface modification strategies.

One alternative approach involves the use of low-temperature plasma (LTP, also known as cold plasma), a partially ionized gas with a variety of electrons, ions, free radicals, and excited atoms and molecules 25 . The interactions of the plasma with the surface of polymeric materials can result in major improvements in wettability and surface charge, changes in surface topography, and can impart antimicrobial activity [26] [27] [28] . LTP treatment can be performed at atmospheric or low pressure (APP, LPP, respectively). APP treatment requires the application of higher voltages for gas breakdown and tends to result in more heating, due to enhanced collisions between electrons and gas molecules 29 . Typically, APP modification is carried out using a nozzle system and to treat large surfaces, a large number of nozzles is required. In contrast, LPP is carried out in vacuum chambers which limit the size that can be modified at one time but improves the plasma distribution, ensuring more even exposure of the surface of the sample 30 . Therefore, the LPP is used commonly for biomedical applications 26, 31 .

Importantly, LPP processes do not require any solvents and do not generate any waste, making them inherently "green" 32 .

The LPP process can use several different inert or reactive gases, which affect the relative amounts and types of surface functional groups formed, such as -OH, -CHO, -COOH, or -NH2. Among the inert gases, argon is the most common 30 , because it has good thermal conductivity, a low electron affinity, a relatively high mass, low ionization energy, and a low rate of electrode erosion 33 Ultimately, the developed filters could play a valuable role as part of protective measures during the current SARS-CoV-2 and any future airborne pandemics.

Optimization of BC-bM preparation process. Fig. 1 ).

These results led us to develop a two-step homogenization and lyophilization process that yielded three-dimensional, sponge-like BC-bMs ( Fig. 1, Supplementary Fig. 1 ).

We optimized the production process of BC-bMs in terms of airflow resistance, the key parameter for use in respiratory PPE. We tested the effect of process parameters: pulp volume, pulp density, and freezing temperature. A summary of the influence of these parameters on airflow resistance is presented in Table 1 , while Fig. 1 

showing macroscopic homogeneity and porosity of representative BC-bMs. Based on airflow resistance measurements and macro-morphological appearance, we selected the following process parameters as optimal: 1:1 mass ratio of BC pulp to water, a volume of 80 mL (in a 144 cm 2 square petri dish), and freezing temperature of -18 °C (indicated in bold in Table 1 and in green on Fig. 1 ). Using lower pulp density, lower pulp volume, or lower temperature resulted in materials that met airflow resistance requirements but had irregular and heterogeneous macromorphology ( Fig. 1 ). Images were taken using stereoscopic microscope.

Optimization of BC-bM functionalization process. Once we established and optimized the process for obtaining homogenous BC-bM that fulfilled the requirements of EN 13274- 3:2008 in terms of airflow resistance, we proceeded to develop an LPP-Ar functionalization process aimed at improving antibacterial and antiviral properties. We varied the treatment time from 1 to 30 min, to optimize the LPP-Ar functionalization process.

First, we used ATR-FTIR to assess changes in the chemical composition of LPP-Ar-BC-bMs depending on the duration of the functionalization process. The ATR-FTIR spectrum of BC-bM served as a control (Fig. 2a) . In the first part of the spectrum (from 2600 cm -1 to 3600 cm -1 ), characteristic bands for OH of hydrogen bonds at 3334 cm -1 and CH at 2895 cm -1 were observed. The second region of spectra (from 1800 cm -1 to 800 cm -1 ) showed signals from adsorbed water at wavenumber 1648 cm -1 , CH2 of C-6 at 1427 cm 1 , COH in-plane from C-2 and C-3 at 1134 cm -1 , OC of β-glycosyl linkage at 1161 cm 1 , CO at C-6 at 1030 cm -1 and COC of β-glycosylic linkage characteristic for amorphous BC fraction at 897 cm -1 . We repeated the ATR-FTIR measurements after 1, 2, and 3 months of storage of LPP-Ar-BC-bMs at room temperature in a desiccator. We did not observe any significant reduction in the intensity of the absorption band at 1720 cm -1 during storage, as compared to the initial value immediately after LPP-Ar treatment ( Supplementary Fig. 3 Based on the results obtained using ATR-FTIR, XRD, and TGA, it was concluded that 10 min was an optimal time for LPP-Ar treatment. Therefore, further analyses, presented below, were carried out only for LPP-Ar-BC-bMs functionalized for 10 min. indicate statistically significant differences (p<0.05).

In order to assess the effect of the functionalization process on the microstructure of the LPP-Ar-BC-bM functionalized for 10 min, we used SEM analysis. A clear effect of LPP-Ar treatment on the surface morphology (roughness and porosity) was observed (Fig. 4a, b, Supplementary Fig. 4 ). Both LPP-Ar-BC-bM and control BC-bM had differentiated pore structures, with both macropores (>100 µm) and micropores (<100 µm) visible. However, LPP-Ar-BC-bM was characterized by a higher degree of porosity compared to control BC-bM ( Fig. 4a, b, Supplementary Fig. 4 , Supplementary Fig. 5 ). The maximum, minimum, as well as average pore diameter of LPP-Ar-BC-bM were all significantly higher, as compared to the control BC-bM ( Fig. 4c ). At the same time, the variability in the size of the pores was also much greater in the case of LPP-Ar-BC-bM. well as strong antiviral activity against bacteriophage Φ6 (Fig. 5a) , an enveloped bacteriophage that can be used as a model surrogate for studying the surface and air survival of pathogenic viruses, including SARS-CoV-2 36 . In both cases, we observed a >99% reduction in cell/phage viability, as compared to the control BC-bM. Further, we repeated the tests with LPP-Ar-BC-bMs after 1, 2, and 3 months of storage and saw no reduction in antibacterial and antiviral activity (Fig. 5b) .

Additionally, we also used SEM to check for the presence of Φ6 phages on the surface of LPP-Ar-BC-bM and BC-bM. First, we confirmed that we were able to visualize phage Φ6 particles with the expected diameter (Fig. 5c) . However, while we were able to observe phage particles on the control BC-bM, we did not note the presence of phages on the surface of LPP-Ar-BC-bM, which is consistent with the strong antiviral effect observed (Fig. 5d,e) . 6a ). There was no difference in viability between the LPP-Ar-BC-bM and control BC-bM. The morphology of L929 cells was not altered (Fig. 6c) .

Likewise, the direct contact assay, where discs of samples were placed directly on top of L929 fibroblast cells for 24 h, also did not show any difference between LPP-Ar-BC-bM and control BC-bM (Fig. 6b) . We did not see altered morphology or reduced cell numbers when imaging wells without removing discs for the resazurin viability assay. Both directly under the discs and at the edges of the discs, cells were normal and dense (Fig. 6d) . Evaluation of adsorption capacity of LPP-Ar-BC-bM. While our goal was to use LPP-Ar treatment to obtain the materials with antimicrobial and antiviral activity, we wanted to ensure that LPP-Ar treatment, which affected microstructure and porosity, did not reduce the ability of the materials to trap particles via adsorption. In fact, the results showed a modest increase in bacterial adsorption capacity for LPP-Ar-BC-bM, as compared to the control BC-bM (Table 2) .

Meanwhile, for the case of our model viral pathogen, phage Φ6, the LPP-Ar-BC-bM exhibited nearly 2-fold greater adsorption capacity, as compared to the control BC-bM. 

in accordance with EN 14683+AC: 2019-09. Our results indicated that 1 layer of LPP-Ar-BC-bM provides on average ~80% bacterial filtration efficiency (BFE), but by arranging them in three layers, it was possible to ensure > 99% of BFE (Table 3) . Importantly, for the case of phage Φ6, intended to model respiratory pathogens like SARS-CoV-2, just one layer of LPP-Ar-BC-bM ensured VFE above 99%.

Importantly, it was also confirmed that two or three layers of LPP-Ar-BC-bM still met the airflow resistance requirements for P1, P2, or P3 class filters according to European standards

Breathing Resistance (Table 3) . User experience testing of prototype masks with LPP-Ar-BC-bM filter. As a final proofof-concept, to demonstrate the potential of the developed functionalized materials to be used in PPE, we prepared 80 NanoBioCell masks and had 80 volunteer medical professionals wear them for 3 h. The participants then completed a user experience survey. In terms of comfort and fit, over all categories, only ~1% of participants rated the NanoBioCell masks as "poor", with the vast majority (~60%) scoring the masks as "good" (Fig. 7a, Supplementary Fig. 6 ).

Encouragingly, more participants (~25%) rated the prototype masks as "very good" as compared to "average" (~14%). In terms of moisture absorption, 81% of respondents described the NanoBioCell as "dry" after 3 h of use and only ~1% rated it as "wet" (Fig. 7b) . The results were presented as % of responses against the number of respondents (n=80).

We hypothesized that the BC is well suited to function as a biobased, biodegradable filter material thanks to its unique nanoscale fibril morphology. Further, we aimed to use the LPP-Ar to improve antimicrobial and antiviral properties, without the need for harsh solvents or generating other waste. Ultimately, our goal was to develop a strategy to obtain biobased, biodegradable filters with excellent filtration efficiency parameters and antibacterial and antiviral properties that could be used in masks and other PPE. In this regard, such filters could act as sustainable, indispensable protective measures during the current SARS-CoV-2 pandemic or any future respiratory pathogen pandemic.

Despite the porous nanostructure of BC, its dense network makes obtaining sufficiently low airflow resistance particularly challenging 16 . In fact, we were not able to obtain sufficiently low airflow resistance values from dried BC pellicles to make them suitable as filters.

Therefore, we decided to explore a homogenization and lyophilization process to obtain BC-bMs. The goal was to reduce the density and thus reduce airflow resistance. We tested different process conditions (BC content in the homogenized pulp and the pulp volume to surface ratio) that yielded materials with different densities and thicknesses. The BC-bMs were then tested for their airflow resistance in accordance with the European standard EN 13274-3:2008 for testing respiratory PPE. As a result, we were able to optimize process parameters to obtain excellent airflow resistance results while maintaining the homogeneous, consistent, threedimensional structure of the BC-bMs.

Next, we aimed to obtain the antibacterial and antiviral properties of our BC-bMs using LPP-Ar. In contrast to various "wet" textile processing methods, LPP treatment is a solventfree ("dry") process that generates no waste, making it environmentally friendly and "green" 13 .

However, the scope of research into LPP-based modification of BC has thus far been limited to enhancing cell affinity, adhesion, or change wettability [37] [38] [39] [40] [41] .

To guide the optimization of LPP-Ar process conditions, we used ATR-FTIR spectroscopy. 27, 34, 35, 48 . Although low-temperature plasma (LTP) is a method commonly used for polymer surface modification, the mechanism responsible for antimicrobial activity (especially in the case of porous surfaces) remains to be fully elucidated 27 .

LTP treatment may change oxidation, nitration, hydrolyzation, and/or amination of the surface, which can affect both prokaryotic and eukaryotic cell attachment and viability 27, 49 .

In contrast to the well-established antimicrobial effect of LPP treatment, there has been limited research into antiviral activity. A direct antiviral effect of LPP-O2 100% and LPP-Ar 80% + O2 20% has been demonstrated towards bovine viral diarrhea virus and the porcine parvovirus, which are surrogates of human hepatitis C virus and human parvovirus B19, respectively 50,51 , but this makes it a sterilization process, rather than a lasting functionalization.

However, in this work we showed that 10 min of LPP-Ar could yield LPP-Ar-BC-bM with strong antiviral activity: we observed a >99% reduction in the activity of phage Φ6, as compared to BC-bM. Further, the effect was not diminished over 3 months of storage. Phage Φ6 was chosen as a model virus, because it is of comparable size and has a somewhat similar lipid envelope to SARS-CoV-2, which makes it a suitable non-pathogenic surrogate for studying the surface and air survival of pathogenic viruses, including SARS-CoV-2 36 . Using SEM imaging we did not observe phage Φ6 particles on the surface of LPP-Ar-BC-bM, in contrast to the BC-bM. This suggests that direct contact with the surface of LPP-Ar-BC-bM may cause the lipid envelope of phage Φ6 to disintegrate, resulting in phage inactivation 36 .

Taking into consideration the strong antibacterial and antiviral properties of LPP-Ar-BC-bM, we screened for potential cytotoxicity against eukaryotic, mammalian cells using L929 murine fibroblasts. The results of both extract and direct contact assays based on ISO 10993-5

showed no reduction in L929 viability reduction, no changes in cell morphology, and no differences between LPP-Ar-BC-bM and BC-bM. Therefore, we conclude that LPP-Ar treatment did not have any adverse effect on the cytocompatibility of the materials and that the obtained LPP-Ar-BC-bM can be considered non-toxic.

In order to demonstrate that the developed LPP-Ar-BC-bM is suitable for respiratory PPE, such as surgical masks, we tested filtration efficiency in accordance with ISO 14683+AC:2019-09. This standard defines the degree of filtration efficiency required by distinct types of medical face masks and other medical filtration materials. For type I medical masks, the bacterial filtration efficiency (BFE) must be ≥ 95%, while for type II and IIR it must be ≥98%. Our results showed that by using three layers of LPP-Ar-BC-bM, we could obtain a BFE >99% for S. aureus and E. coli. Further, and perhaps more importantly in the context of the present SARS-CoV-2 pandemic, the viral filtration efficiency (VFE) was >99% with single layer LPP-Ar-BC-bM. It is important to note that three layers of LPP-Ar-BC-bM still met the requirements of the EN 13274-3: 2008 standard for airflow resistance (6.0±0.43 mbar resistance at an airflow of 95 L/min). According to EN 13274-3:2008 standard, at an airflow of 95 L/min, the air resistance of respiratory PPE should not exceed 6.1 mbar for a class P1 filter, 6.4 mbar for a class P2 filter (equivalent to a N95 respirator), and 8.2 mbar for class P3 filter.

Overall, we conclude that the high filtration efficiency of our three-layers LPP-Ar-BC-bM filter is the result of the combination of the unique structure of BC-bM and LPP-Ar induced changes in isoelectric charge, polarity, reactivity, and wettability, which increase biomolecule adsorption [52] [53] [54] . It has been previously shown that LTP-modified surfaces have an increased ability to adsorb proteins present in mammal and bacterial cells [55] [56] [57] . In fact, Griffin et al.

confirmed that LPP-Ar surface modification correlated with the higher adsorption of such proteins, as compared to LPP treatment with oxygen or nitrogen 57 . For the case of enveloped viruses like SARS-CoV-2 (and the surrogate we used, phage Φ6) the virus is protected by an outer lipid bilayer with a high density of proteins, such as the crucial spike protein [58] [59] [60] . We hypothesize that it is interactions with these proteins that may explain the high ability of LPP-Ar-BC-bM filters to both adsorb and deactivate viral particles.

As a final proof-of-concept that our filters could be used in PPE filtration devices, we prepared 80 NanoBioCell masks and asked volunteer medical staff to wear them for 3 h. The majority of scores were above the score "average", which indicates that, from a comfort perspective, the NanoBioCell masks were comparable to, or better than, the existing PPE used by the participants. Only ~1% of responders rated the masks as "poor", which may have been the result of imperfect internal quality control, because members of the research team and not a professional company made not just the filters but the entire masks. When combined with the fact that the LPP-Ar-BC-bM filters had excellent BFE and VFE and are fully biobased and biodegradable, these results establish the excellent potential of the developed process and materials to be used for PPE products.

In summary, in the current work, we showed that it is possible to fabricate air filters using bacterial cellulose (BC) by using homogenization and lyophilization. Further, the filters could be functionalized using low-pressure argon plasma (LPP-Ar), which markedly improved antimicrobial and antiviral properties. Such three-layers LPP-Ar-BC-bM filters provided strong antibacterial and antiviral properties, as well as bacterial and viral filtration efficiencies >99%.

At the same time, the three-layers LPP-Ar-BC-bM filters met the airflow resistance requirements of the EN 13274-3: 2008 standard for respiratory PPE. Further, despite the potent antimicrobial and antiviral effect, we did not observe any indication of cytotoxicity caused by the LPP-Ar-BC-bM. Importantly, by combining the biotechnological BC production with "green" low-temperature plasma functionalization, the entire process is environmentally friendly. Our filters do not require the use of any harmful solvents and produce no hazardous waste. Finally, as a proof-of-concept, we were able to prepare 80 NanoBioCell masks and ~85%

of volunteer medical staff assessed them as "good" or "very good" in terms of comfort. As a result, we conclude that our LPP-Ar-BC-bM filters can be used in PPE face masks and respirators, offering a biobased, biodegradable, sustainable and safe alternative to existing materials. Further, we believe that with scale-up, the developed technology could be used for large indoor air filtration systems, such as those in hospitals or schools.

Preparation of BC-based material. In the first stage, BC was biosynthesized using a reference strain of In the next stage, the purified BC pellicles were homogenized using a blender (Perfectmix + BL811D38, Tefal, France) with the addition of distilled water at a weight ratio of 1:1, 1:2, or 2:1. Once homogeneous BC pulp was obtained, it was poured into square Petri dishes (120 x 120 x 17 mm) at a volume of 60 mL, 80 mL, or 100 mL. The pulp was then frozen at -18 °C or -80 °C for 24 h and then lyophilized at -60 °C and 0.1 mBar (Alpha 1-2

Ldplus, Christ, Germany) to yield BC-based materials (referred to as "BC-bMs") ( Fig. 8) . Fig. 7) . The tests were conducted at airflow rates of 30 L/min and 95 L/min, corresponding to a respiratory minute volume during moderate and strenuous activity.

The BC-bMs were functionalized with a low-pressure argon plasma (LPP-Ar) using HPT-100 Benchtop Plasma Treater (Henniker Plasma, UK). The gas flow and power were held constant at 10 sccm (chamber pressure ~0.6 mbar) and 100% power (100 W). These process parameters were selected in cooperation with a technical support staff of Henniker Plasma and yielded stable, well-distributed plasmas within the chamber.

The treatment time was varied from 1 to 30 min. After LPP-Ar treatment, the functionalized BC-bMs (referred to as "LPP-Ar-BC-bMs") were stored at room temperature in a desiccator until further analysis.

Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) was used to characterize functional groups and detect changes in chemical composition in LPP-Ar-BC-bMs. The measurements were performed using ALPHA FT-IR Spectrometer (Bruker Co., Germany) with a DTGS detector and the platinum-ATR-sampling module with a robust diamond crystal and variable angle incidence beam. For each sample, 32 scans at 2 cm -1 resolution were recorded over the spectral range of 4000 -400 cm -1 . The spectra were processed by baseline correction, smoothed with a polynomial Savitzky-Golay filter, and normalized to band area at 1161 cm -1 using the SpectraGryph 1.2 software package. Processed spectra were then analyzed using twodimensional correlation spectroscopy in OriginPro2021 software. The BC-bM was used as a control. Additionally, ATR-FTIR was used to assess the stability of functionalization after 1, 2, and 3 months of storage of LPP-Ar-BC-bMs at room temperature in a desiccator.

X-ray diffraction analysis. X-ray diffraction analysis (XRD) using a D5005 X-ray diffractometer (Bruker Siemens, USA) was used to assess the crystallinity of LPP-Ar-BC-bMs. A diffraction angle 2 θ was measured from 5° to 70° with a step size of 0.04° using Cu-kα radiation at 40 kV and 40 mA. Crystallinity (%) was calculated by dividing the area of the crystalline peaks by the total area under the curve from 2θ 5° to 30° 62 , using the Diffrac.eva software by Bruker (USA). The BC-bM was used as a control.

Thermogravimetric analysis. The thermal stability of LPP-Ar-BC-bMs was evaluated using a Perkin Elmer TGA 8000 thermogravimetric analysis (TGA) system. Samples were conditioned at room temperature before measurements without a pre-heating cycle, to obtain water evaporation data. Then, during the experiment, samples (~6 mg) were placed in ceramic sample pans and were heated from 30 °C to 700 °C, at a heating rate of 10 °C/min under nitrogen atmosphere. The BC-bM was used as a control. 

Original datasets discussed in this publication have been deposited with link to Figshare database (https://figshare.com/articles/dataset/Argon_plasma-modified_bacterial_cellulose_filters_for_protection_against_respiratory_pathogens/19615236).

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This research was funded by the Regional Operational Program of the West Pomeranian Voivodeship, Grant No.Proto_lab/ K1/2020/U/11 and Proto_lab/K2/2021/U/7. We would like to thank Grace Law, Henniker Plasma, for technical advice and support regarding low pressure argon plasma parameters. We would also like to thank Xymena Stachurska for help in phage Φ6 lysate preparation.

The authors declare no competing interests.