International Journal of Environmental Research and Public Health

Article Evaluation of the Inactivation Ability of Chitosan-Coated Antimicrobial Filters

Ying-Fang Hsu 1, Chi-Yu Chuang 2,* and Shinhao Yang 1,*

1 Environmental Sustainability Lab, Center for General Education, CTBC Business School, No. 600, Section 3, Taijiang Boulevard, Annan District, Tainan 709, Taiwan; [email protected] 2 Department of Occupational Safety and Health, Chang Jung Christian University, No. 1, Changda Road., Gueiren District, Tainan City 711, Taiwan * Correspondence: [email protected] (C.-Y.C.); [email protected] (S.Y.); Tel.: +886-6-2785123-7503 (C.-Y.C.); +886-6-2873198 (S.Y.)

Abstract: This work considers the enhancement of indoor bioaerosol removal efficiency by liquid coating of the antimicrobial agent chitosan onto polypropylene fibrous filters (CCFs). Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) were chosen as the tested . The results revealed that 2.5% (w/w) of CCFs have significantly higher bioaerosol survival capability (23% and 34% of E. coli and B. subtilis, respectively), compared to an untreated filter (65% and 64% for E. coli and B. subtilis, respectively). Increasing face velocity and relative during operating CCFs could reduce the bioaerosol removal capability. The regression analysis of the experimental findings demonstrated that the higher coating concentration of chitosan had the most positive influence on bioaerosol removal, while the face velocity and relative humidity had a negative influence, but a milder effect   was observed (R2 = 0.83 and 0.81 for E. coli and B. subtilis bioaerosols, respectively). A CCF-loaded air-cleaning device was tested in a real indoor environment and resulted in 80.1% bioaerosol removal Citation: Hsu, Y.-F.; Chuang, C.-Y.; within 3 h of operating, which suggests that the chitosan-coated filter has the potential for further Yang, S. Evaluation of the Bioaerosol Inactivation Ability of application in improving in the future. Chitosan-Coated Antimicrobial Filters. Int. J. Environ. Res. Public Keywords: chitosan-coated filters; survival; bioaerosols; antimicrobial agent Health 2021, 18, 7183. https:// doi.org/10.3390/ijerph18137183

Academic Editors: Daniela Varrica 1. Introduction and Atin Adhikari The investigation displayed that modern people spend an average of 87.2% of their time indoors. Thus, indoor air quality is a significant environmental and health issue [1]. Received: 25 March 2021 Among the indoor contamination sources, airborne , the major composition Accepted: 2 July 2021 of bioaerosols, can cause various respiratory problems, allergic syndromes, and infectious Published: 5 July 2021 , as well as have a serious impact on indoor air quality [2,3]. Therefore, many air-cleaning techniques, including ozone releasing, photocatalytic oxidation, ultraviolet ger- Publisher’s Note: MDPI stays neutral micidal irradiation, plasma neutralization, and disinfectant spraying, have been developed with regard to jurisdictional claims in for inactivating indoor airborne microbial contamination. published maps and institutional affil- Li and Wang [4] investigated the inactivating ability of the ozone on bioaerosols. iations. Their results demonstrated that when the ozone concentration was increased to 10 ppm, the germicidal efficiency toward the and the fungi bioaerosols began to become obvious. Lin and Li [5] examined the effectiveness of titanium dioxide photocatalyst filters under a wavelength of 365 nm ultraviolet light in controlling bioaerosols. They Copyright: © 2021 by the authors. found that the effect of photocatalytic oxidation on bioaerosol removal was weak. Lin and Licensee MDPI, Basel, Switzerland. Li [6] employed ultraviolet germicidal irradiation to remove indoor bacteria and fungi This article is an open access article bioaerosols. Their findings indicated that ultraviolet germicidal irradiation has the best distributed under the terms and removal efficiency for Escherichia coli (E. coli) bioaerosols and the poorest ability to control conditions of the Creative Commons spores of Penicillium citrinum. When the ultraviolet germicidal irradiation was applied Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ to the indoor environment for 10 min, the concentration of environmental bioaerosols 4.0/). decreased greatly. However, the ultraviolet germicidal irradiation may also generate a

Int. J. Environ. Res. Public Health 2021, 18, 7183. https://doi.org/10.3390/ijerph18137183 https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2021, 18, 7183 2 of 11

secondary pollutant (ozone and nitrogen oxides) and subsequently cause the air quality to deteriorate. Yang et al. [7] investigated the ability of a carbon nanotube (CNT) corona discharge plasma system to inactivate bioaerosols. The inactivation efficiencies of E. coli, B. subtilis, and λ bioaerosols using CNT corona discharge plasma were as high as 93%, 88%, and 81%, respectively. Chuang et al. [8] indicated that spraying electrolyzed water containing as high as 200 ppm free available chlorine was revealed to be significantly effective in the inactivation of bacterial airborne contamination. Although all of the above-mentioned available air-cleaning technologies are effective in bioaerosol inactivation, environmentally friendly (such as the minimum disinfectant residual) standards and personal safety requirements cannot be fulfilled. On the other hand, filtration, as the most traditional air-cleaning technology, has the strengths of being non-residual and safe for humans. In addition, some studies are pursuing the improved efficiency of non-woven air filters in capturing bioaerosols. Shi et al. [9] indicated that the polypropylene electret filter is highly efficient in removing bacterial aerosol from air. In Yang’s study [10], an air filter was pretreated with electric charge and surfactant to increase its capacity to capture E. coli and yeast bioaerosols. In addition, Yang’s study indicated that indoor environment parameters, such as face velocity and relative humidity, influence the removing efficiency of bioaerosols due to shorting retention time and surfactant activity, respectively. Recently, air-filtering media treated with natural antimicrobial agents have been in high demand as they can achieve desirable collection efficiency and self-deactivate the viability of bioaerosols, eventually resulting in a reduced biocontamination risk in the indoor environment. Hereby, this work developed an easily prepared, low-cost, and antimicrobial filter for inactivating indoor bacterial bioaerosols. Chitosan is a natural biopolymer composed of two chemical elements, randomly distributed β-(1→4)-linked D-glucosamine and N-acetyl- D-glucosamine. Chitosan has been used in a variety of industries, including medical, food, textiles, cosmetics, agriculture, and chemical engineering. Many studies have indicated that chitosan coated into textile fibers or added into liquid is an acceptable antimicrobial agent, which has a significant ability to inactivate microorganisms [11–18]. Ozden and Basal (2017) applied polyamide 6/chitosan nanofiber-coated on HEPA filter for controlling bioaerosols [19]. Chen et al. (2019) used zero-valent nano-silver/Ti02-chitosan composite filters to remove hospital indoor bioaerosols [20]. Mishra et al. (2021) applied lemongrass essential oil loaded on chitosan nanocellulose for inactivating bioaerosols [21]. These above studies used chitosan’s antibacterial ability to inactivate bioaerosols. However, the applications of chitosan in these studies were coating chitosan on a high-efficiency HEPA filter or combining chitosan with multiple antibacterial ingredients on a filter. Therefore, this work aimed to develop an antimicrobial filter by using the low- efficiency filter coated with the antimicrobial agent chitosan, characterizing its overall removal performance in removing biological and non-biological aerosols. The addition of the biological inactivating effects of a chitosan coating were clarified by comparing the overall removal performance among biological and non-biological aerosols (tested and expressed as penetration rate). First, this work utilized standard, non-biological la- tex aerosol to test the newly developed chitosan-coated filters (CCFs) to evaluate their physical aerosol penetration rate. Second, based on the physical aerosol penetration rate of non-biological aerosols, E. coli and Bacillus. subtilis (B. subtilis) were then selected as the analyzed bioaerosols to elucidate their biological inactivating ability, examined by combining physical penetration with survival conditions through CCFs. Two essential environmental factors that majorly affect filtering performance, relative humidity and face velocity, were included in the study.

2. Materials and Methods 2.1. Filter Media The characteristics of the non-antibacterial original filter and CCFs are both presented in Table1. A commercial single-layer polypropylene (PP) fibrous air filter was purchased as Int. J. Environ. Res. Public Health 2021, 18, 7183 3 of 11

the original untreated media in the filter (KNH Enterprise Co., Ltd., Tainan, Taiwan). The antibacterial CCFs were prepared by using original polypropylene fibrous filters coated with chitosan at various concentrations (1.0%, 1.5%, and 2.5%, w/w). The chitosan-coating solutions were made by dissolving chitosan (Merck & Co., Inc., Kenilworth, NJ, USA) in pure water. The coating procedure of the CCFs is shaking (50 rpm) the original filter media with chitosan solution for 1 min (under 30 ◦C) to sufficiently coat the chitosan on the filter fiber. The chitosan solution was mixed completely and the time the filter soaked in the solution was long enough for chitosan coating on the fiber surface. After the coating procedure, the CCFs were dried in a condition of 105 ◦C for 12 h. The uniformity of CCFs was very good in this study. According to the observation of scanning electron microscope (SEM), the coated fiber diameters were nearly the same, and the chitosan was coated on fiber surface uniformly.

Table 1. Fiber characteristics of original filters and CCFs.

Measured Measured Measured Weight of Mean Fiber Mean Filter Type Material Pa@10 cm/s Filter Diameter a Thickness b (g/m2) (µm) (cm) Untreated Polypropylene 60.5 ± 0.1 10.1 ± 0.3 0.10 ± 0.01 5.1 ± 0.1 Polypropylene/ 1.0% CCF 66.3 ± 0.2 10.2 ± 0.5 0.11 ± 0.00 5.1 ± 0.2 Chitosan Polypropylene/ 1.5% CCF 70.2 ± 0.3 10.3 ± 0.3 0.10 ± 0.01 5.1 ± 0.1 Chitosan Polypropylene/ 2.5% CCF 75.3 ± 0.2 10.2 ± 0.1 0.10 ± 0.01 5.1 ± 0.1 Chitosan

a Diameter was measured by SEM experiments. b Filter thickness was measured by digital vernier; the mean fiber thickness was averaged by triple tests.

2.2. Tested Bioaerosols According to the previous studies, E. coli and B. subtilis were chosen as the analyzed bioaerosols for indoor bioaerosol inactivating tests [22,23]. E. coli, a Gram-negative bacterial strain, was selected to represent human activity-related indoor environmental bacterial species. On the other hand, B. subtilis, a Gram-positive endospore-forming bacterial strain, commonly found in soil and rotten plants, was selected to represent bio-contaminants from the atmospheric environment in this study. Following previous laboratory-based bioaerosol studies [6–8,24], two different types of bioaerosols were analyzed individually to understand their specific survival capability against antimicrobial agents using the culture method. Accordingly, the vegetative cells of E. coli (Bioresource Collection and Research Center in Taiwan, BCRC 10675) and B. subtilis (Culture Collection & Research Center in Taiwan, CCRC 12145) were purchased and activated for the preparation of bioaerosols based on our previous study [7]. Three E. coli and three B. subtilis colonies were transferred from the individual pure culture agar plate and cultivated into a conical flask containing 30 mL of tryptic soy broth (TSB, Bacto™). The transferred colonies in the TSB were cultivated under a shaking condition of 85 rpm, for 16–24 h at 37 ◦C, for enrichment. After the enrichment, the TSB solution was centrifuged under the condition of 2500 rpm for 5 min, and the resulting supernatant was removed. Then, the E. coli and B. subtilis sediments were re-suspended by adding 30 mL of phosphate buffered saline solution (PBS, pH 7.2). These processes were repeated twice to remove TSB medium. Finally, this PBS solution was used for bioaerosol generation. The initial concentration of both bacterial strains in the solution was determined as 105 CFU/mL with serial dilutions. Then, the bacterial solution was loaded to the Collison three-jet nebulizer (BGI Inc., Waltham, MA, USA) and included in the experimental setup (described in Section 2.4). When supplied with 20 psig clean and compressed air, the nebulizer generates E. coli and B. subtilis bioaerosol streams in a range of 4 to 8 × 104 CFU/m3. Int. J. Environ. Res. Public Health 2021, 18, 7183 4 of 11

2.3. Tested Non-Biological Aerosols This work applied a monodisperse polystyrene latex (PSL, Duke, CA, USA) sphere particle (geometric diameter: 0.9 µm; GSD: 1.02; presents similar aerodynamic behavior with E.coli and B. subtilis bioaerosols) as the testing aerosol to understand the efficiency with which aerosols penetrate the CCF filters according to previous studies [25,26]. The PSL aerosols were generated using the Collison three-jet nebulizer (BGI Inc., Waltham, MA, USA), and measured by an Aerodynamic Particle Sizer (APS, model 3320, TSI Inc., Shoreview, MN, USA).

Int. J. Environ. Res. Public Health 2021, 182.4., x Aerosol Experimental Setup 5 of 12 Figure1 schematically describes the aerosol experimental setup. This work utilized to test the physical removal efficiency and biological inactivating ability through the CCFs. The aerosol experimental setup includes the Collison three-jet nebulizer, the airflow mixing an Andersen one-stage sampler. The bioaerosol collection flow rate of the sampler was set column, the testing filter holder (100 mm diameter, to support the CCFs filter and maintain at 28.3 lpm based on the operation manual. Bacterial bioaerosols were collected on tryptic its integrity during aerosols stream passing), the Anderson one-stage impaction sampler soy agar (TSA, Difco Laboratories, Detroit) and cultured at 37 °C for 24 h. (Andersen Samplers, Inc., Atlanta, GA, USA), APS, and flowmeters.

Compressed Air Compressed Air HEPA Compressed Air

HEPA Air Supply Flow Nebulizer Meter

Saturator KR85 Diffusion Flow Dryer Meter

Mixing Column

Filter Holder

APS

Andersen Pump One-stage Sampler FigureFigure 1. Schematic 1. Schematic diagram diagram of ofthe the experimental experimental system. system.

3. ResultsThe and analyzed Discussion aerosols (namely, non-biological and biological) were aerosolized by the Collison three-jet nebulizer (BGI Inc., Waltham, MA, USA) and dried by passing 3.1. Non-Biological Aerosol Removal through CCFs them through a diffusion dryer as a stream. The dried aerosol stream was be neutralized andFigure checked 2 shows by using the P aStd Kr-85 of 0.9 radioactive μm PSL aerosols source through (model the 3077, uncoated TSI Inc.) single-layer and an aerosol PP materialelectrometer filter, and (model the 3068,1.0%, TSI1.5%, Inc.). and The 2.5% analyzed CCFs, at aerosols face velocities were mixed of 10, with 20, and diluted 30 cm/s clean (RHair 30%), and flowed respectively. into the The mixing results column. showed In the this aerosol work, threepenetrations face velocities through (10, the 20, un- and treated30 cm/s filter,) through and the the 1.0%, tested 1.5%, filter and were 2.5% selected. CCFs at Thea face face velocity velocity of through10 cm/s were the tested approx- filter imatelywas controlled 63.1%, 58.9%, by the 55.3%, diluted and clean 51.1%, air respectively. flow (face velocities The non-biological, = Q/A, Q equal standard to analyzed PSL aerosolaerosols penetration flow + diluted through clean the three air flow; CCFs A was equal lower to tested than that filter of area).the original In this uncoated work, we filter,selected up to three 12% relative(from 63.1% humidity to 51.1%, (RH, 30%,under 50%, 10 cm/s and 70%)face conditionsvelocity). Increasing to simulate the the face ideal velocityindoor of environment the filters, on (RH the range:contrary, 30–50%, minimized according the difference to U.S. EPA between indoor the aerosol control). pen- A etrationhigh humidity rates (from condition 68.2% to (RH 59.1%, 70%) under was also 30 cm/s). included These in this chitosan-coating work to simulate addition the indoor ef- fectscondition also found in a subtropicalthat as the coated zone. The chitosan RH of concentration the analyzed aerosolincreased, flow the in theaerosol mixing penetra- column tionwas (PStd controlled) decreased. by changingPretreating the the ratio filter of with theflow chitosan rate could of humidified increase gasthe streamthickness generated of the filterby theandwater cause vapora higher saturator interception and dry effect gas toward stream. aerosols. The RHof However, analyzed in aerosol this work, flow the were observedsampled result using shows a hygrometer that pretreating (Hygromer the filter A2, Rotronic did not significantly Inc., Hauppauge, alter the NY, mechanical USA). characteristicsThe physical of the aerosolfilter fiber penetration (shown as efficiency Table 1). ofTherefore, the testing this filter indicates was evaluated that when by the ana- filterlyzing was the coated PSL aerosol.with chitosan, The physical the fiber aerosol surface penetration properties efficiency were altered (PStd, %)and was resulted defined in as an enhancement of the electrostatic aerosol-capturing forces, which resulted in a strong molecular polarity and positively-charged NH3+ group of chitosan. As a result, the phys- ical collection efficiency (i.e., penetration prevention) of aerosols was increased. Addition- ally, this electrostatic enhancing effect was reduced when face velocity was increased. A similar finding was also observed in other studies, which applied polymers to air filters [27,28].

Int. J. Environ. Res. Public Health 2021, 18, 7183 5 of 11

the ratio of Cwith/Cwithout, where Cwith represents the physical aerosol counting concen- trations (numbers/m3) sampled from the downstream of the filter holder installed with a CCF filter using an APS. Cwithout represents the physical aerosol counting concentrations (numbers/m3) sampled from the downstream of the filter holder without a CCF filter measured by APS. Considering the biological inactivating effects of chitosan coating, the filter was evalu- ated for its survival recovery of bioaerosols (R%, REc indicates the survival of E.coli and SBs indicates the survival of B. subtilis), which was calculated in the ratio of Swith/Swithout, 3 where Swith represents the bioaerosol culturable concentrations (CFU/m ) sampled from the downstream of the filter holder installed with a CCF filters using an Andersen one-stage 3 impaction sampler. Swithout represents the culturable concentrations (CFU/m ) sampled from the downstream of the filter holder without a CCF filter and measured using an Andersen one-stage sampler. The bioaerosol collection flow rate of the sampler was set at 28.3 lpm based on the operation manual. Bacterial bioaerosols were collected on tryptic soy agar (TSA, Difco Laboratories, Detroit) and cultured at 37 ◦C for 24 h.

3. Results and Discussion 3.1. Non-Biological Aerosol Removal through CCFs

Figure2 shows the P Std of 0.9 µm PSL aerosols through the uncoated single-layer PP material filter, and the 1.0%, 1.5%, and 2.5% CCFs, at face velocities of 10, 20, and 30 cm/s (RH 30%), respectively. The results showed the aerosol penetrations through the untreated filter, and the 1.0%, 1.5%, and 2.5% CCFs at a face velocity of 10 cm/s were approximately 63.1%, 58.9%, 55.3%, and 51.1%, respectively. The non-biological, standard PSL aerosol penetration through the three CCFs was lower than that of the original uncoated filter, up to 12% (from 63.1% to 51.1%, under 10 cm/s face velocity). Increasing the face velocity of the filters, on the contrary, minimized the difference between the aerosol penetration rates (from 68.2% to 59.1%, under 30 cm/s). These chitosan-coating addition effects also found that as the coated chitosan concentration increased, the aerosol penetration (PStd) decreased. Pretreating the filter with chitosan could increase the thickness of the filter and cause a higher interception effect toward aerosols. However, in this work, the observed result shows that pretreating the filter did not significantly alter the mechanical characteristics of the filter fiber (shown as Table1). Therefore, this indicates that when the filter was coated with chitosan, the fiber surface properties were altered and resulted in an enhancement of the electrostatic aerosol-capturing forces, which resulted in a strong molecular polarity and positively-charged NH3+ group of chitosan. As a result, the physical collection efficiency Int. J. Environ. Res. Public Health 2021, 18, x 6 of 12 (i.e., penetration prevention) of aerosols was increased. Additionally, this electrostatic enhancing effect was reduced when face velocity was increased. A similar finding was also observed in other studies, which applied polymers to air filters [27,28].

Figure 2. Penetration through the chitosan pretreated and untreated filters for 0.9-µm PSL aerosols. Figure 2. Penetration through the chitosan pretreated and untreated filters for 0.9-μm PSL aero- sols.

3.2. Survival Evaluation of Bacterial Bioaerosols through CCFs

Figure 3 shows the survival of E. coli bioaerosols (REC) through the untreated filter, and the 1.0%, 1.5%, and 2.5% CCFs (under face velocity of 10 cm/s and RH of 30%). These data indicate that the REC of E. coli bioaerosols through the untreated filter, and the 1.0%, 1.5%, and 2.5% CCFs were approximately 65%, 46%, 38%, and 23%, respectively. Com- paring these data with the results of PStd of the PSL aerosol through these testing filters, it was found that the survival of the E. coli bioaerosol and the PSL aerosol through the un- treated filter were similar (PStd = 63.1% and REc = 65%, under face velocity 10 m/s). However, the survival of the E. coli bioaerosol through the CCFs was much lower than that through the PSL aerosol, indicating that chitosan has an additional antibacterial effect on the E. coli bioaerosol. The penetration (PStd) of the PSL aerosol through the CCFs revealed that the electro- static force increased when the filter was coated with chitosan. In the filtration of the E. coli bioaerosol using CCFs, the enhanced electrostatic force and antimicrobial effects were added to the original capture force from the untreated filter. Accordingly, 28% of the var- iations in the survival (REc = 23%, 2.5% CCFs under face velocity 10 m/s) of the E. coli bioaerosol and the PSL aerosol penetration (PStd = 51.1%, 2.5% CCFs under face velocity 10 m/s) through the CCFs were attributed to the antibacterial effect of the filter pretreated with chitosan. In addition, the differences between REc of the E. coli bioaerosol and the PSL aerosol penetration (PStd) through the 1.0%, 1.5%, and 2.5% CCFs were approximately 13%, 17%, and 28%, respectively. The experimental results reveal that a higher concentration of CCFs corresponds to a decrease in E. coli bioaerosol survival, resulting in a larger differ- ence between E. coli (REC = 46% and 23% on 1.0% and 2.5% CCFs, respectively) and PSL (PStd = 58.9% and 51.1% on 1.0% and 2.5% CCFs, respectively). These findings could be realized because the filter coated with a higher concentration of chitosan has a stronger antimicrobial effect against E. coli bioaerosols. Figure 4 shows the survival (RBs) of B. subtilis bioaerosols through the testing filters under a face velocity of 10 cm/s and RH of 30%. The data reveal that the RBs of B. subtilis bioaerosols through the untreated filter, and the 1.0%, 1.5%, and 2.5% CCFs were approx- imately 64%, 50%, 46%, and 34%, respectively. The survival of the B. subtilis bioaerosol and the PSL aerosol through the untreated filter were similar (PStd = 63.1% and RBs = 64%, under face velocity 10 m/s). However, the B. subtilis bioaerosols through the CCFs were much lower RBs than those of the PSL aerosol (RBs = 34% and PStd = 51%, 2.5% CCFs under face velocity 10 m/s). This demonstrates that chitosan has a biological inactivating ability against the B. subtilis bioaerosol. The differences between the RBs% of the B. subtilis bioaer- osol and the PSL aerosol penetration (PStd) through the 1.0%, 1.5%, and 2.5% CCFs were approximately 9%, 10%, and 17%, respectively. It can be understood that variations in the survival of the B. subtilis bioaerosol (RBs) and the PSL aerosol penetration (PStd) through

Int. J. Environ. Res. Public Health 2021, 18, x 7 of 12

Int. J. Environ. Res. Public Health 2021, 18, 7183 6 of 11 the CCFs were attributed to the biological inactivating effect of the filter coated with chi- tosan. These findings, showing a similar trend to the E. coli bioaerosol tests, reveal that a filter coated with a higher concentration of chitosan results in a greater antimicrobial effect 3.2.against Survival B. subtilis Evaluation bioaerosols. of Bacterial Bioaerosols through CCFs FigureFurthermore,3 shows these the survival CCFs had of a E.higher coli bioaerosols ability to inactive (REC) throughE. coli bioaerosols the untreated (REc = filter, 46%, and38%, the and 1.0%, 23% 1.5%,with 1.0%, and 2.5%1.5%, CCFs and 2.5% (under CCFs, face respectively) velocity of 10than cm/s B. subtilis and RH bioaerosols of 30%). These(RBs = data50%,indicate 46%, and that 34% the with REC 1.0%,of E.coli 1.5%,bioaerosols and 2.5% through CCFs, respectively), the untreated mainly filter, and due the to 1.0%,the fact 1.5%, that and Gram-negative 2.5% CCFswere E. coli approximately is a bacterial 65%,strain 46%, that 38%,is sensitive and 23%, to antimicrobial respectively. Comparingagents, while these Gram-positive data with theB. subtilis results is of a PresistantStd of the bacterial PSL aerosol strain through [22,23] with these a testingmainly filters,peptidoglycan-consisted it was found that the cell survival wall structure. of the E. Therefore, coli bioaerosol compared and the with PSL B. aerosol subtilis throughbioaero- thesols, untreated E. coli bioaerosols filter were are similar more (P antimicrobialStd = 63.1% and agent REc sensitive= 65%, under and faceeasily velocity biologically 10 m/s). in- However,activated theand survivalremoved of from the E. indoor coli bioaerosol air by CCFs. through This thefinding CCFs is wasconsistent much lowerwith the than results that throughof another the bioaerosol PSL aerosol, control indicating approach that in chitosan a previous has anstudy additional of Lin and antibacterial Li [6]. effect on the E. coli bioaerosol.

FigureFigure 3.3. SurvivalSurvival ofof E.E. colicoli bioaerosolsbioaerosols throughthrough thethe chitosan-coatedchitosan-coatedand anduntreated untreatedfilters. filters.

The penetration (PStd) of the PSL aerosol through the CCFs revealed that the elec- trostatic force increased when the filter was coated with chitosan. In the filtration of the E. coli bioaerosol using CCFs, the enhanced electrostatic force and antimicrobial effects were added to the original capture force from the untreated filter. Accordingly, 28% of the variations in the survival (REc = 23%, 2.5% CCFs under face velocity 10 m/s) of the E. coli bioaerosol and the PSL aerosol penetration (PStd = 51.1%, 2.5% CCFs under face velocity 10 m/s) through the CCFs were attributed to the antibacterial effect of the filter pretreated with chitosan. In addition, the differences between REc of the E. coli bioaerosol and the PSL aerosol penetration (PStd) through the 1.0%, 1.5%, and 2.5% CCFs were approximately 13%, 17%, and 28%, respectively. The experimental results reveal that a higher concentration of CCFs corresponds to a decrease in E. coli bioaerosol survival, resulting in a larger differ- ence between E. coli (REC = 46% and 23% on 1.0% and 2.5% CCFs, respectively) and PSL (P = 58.9% and 51.1% on 1.0% and 2.5% CCFs, respectively). These findings could be Std realized because the filter coated with a higher concentration of chitosan has a stronger antimicrobialFigure 4. Survival effect of B. against subtilisE. bioaerosols coli bioaerosols. through the chitosan-coated and untreated filters. Figure4 shows the survival (R Bs) of B. subtilis bioaerosols through the testing filters under3.3. Effect a face of Face velocity Velocity of on 10 Bioaerosol cm/s and Survival RH of through 30%. TheCCFs data reveal that the RBs of B. subtilis bioaerosols through the untreated filter, and the 1.0%, 1.5%, and 2.5% CCFs were Figure 3 shows the survival (REc) of the E. coli bioaerosols through the 1.0%, 1.5%, and approximately 64%, 50%, 46%, and 34%, respectively. The survival of the B. subtilis 2.5% CCFs at face velocities of 10, 20, and 30 cm/s (RH 30%), respectively. The REC of the bioaerosolE. coli bioaerosols and the through PSL aerosol the 2.5% through CCF theincreased untreated from filter 23% were to 37% similar as the (P faceStd = velocity 63.1% and R = 64%, under face velocity 10 m/s). However, the B. subtilis bioaerosols through increasedBs from 10 to 30 cm/s. As shown in Figure 4, the survival (RBs) of B. subtilis bioaer- theosols CCFs through were the much 2.5% lower CCFs RBs increasedthan those from of the 34% PSL to aerosol48% as (RtheBs face= 34% velocity and PStd increased= 51%, 2.5% CCFs under face velocity 10 m/s). This demonstrates that chitosan has a biological from 10 to 30 cm/s. The results display that the REc of E. coli through these three CCFs inactivating ability against the B. subtilis bioaerosol. The differences between the R % of increased with the face velocity, which indicates that the biological inactivating abilityBs of the B. subtilis bioaerosol and the PSL aerosol penetration (P ) through the 1.0%, 1.5%, and the CCFs decreased with the increase in face velocity. ThisStd is probably due to the increase 2.5% CCFs were approximately 9%, 10%, and 17%, respectively. It can be understood that in face velocity (mechanical) reducing the residence time associated with bioaerosols variations in the survival of the B. subtilis bioaerosol (RBs) and the PSL aerosol penetration (PStd) through the CCFs were attributed to the biological inactivating effect of the filter coated with chitosan. These findings, showing a similar trend to the E. coli bioaerosol

Int. J. Environ. Res. Public Health 2021, 18, x 7 of 12

the CCFs were attributed to the biological inactivating effect of the filter coated with chi- tosan. These findings, showing a similar trend to the E. coli bioaerosol tests, reveal that a filter coated with a higher concentration of chitosan results in a greater antimicrobial effect against B. subtilis bioaerosols. Furthermore, these CCFs had a higher ability to inactive E. coli bioaerosols (REc = 46%, 38%, and 23% with 1.0%, 1.5%, and 2.5% CCFs, respectively) than B. subtilis bioaerosols (RBs = 50%, 46%, and 34% with 1.0%, 1.5%, and 2.5% CCFs, respectively), mainly due to the fact that Gram-negative E. coli is a bacterial strain that is sensitive to antimicrobial agents, while Gram-positive B. subtilis is a resistant bacterial strain [22,23] with a mainly peptidoglycan-consisted cell wall structure. Therefore, compared with B. subtilis bioaero- sols, E. coli bioaerosols are more antimicrobial agent sensitive and easily biologically in- activated and removed from indoor air by CCFs. This finding is consistent with the results of another bioaerosol control approach in a previous study of Lin and Li [6].

Int. J. Environ. Res. Public Health 2021, 18, 7183 7 of 11

Figuretests, reveal 3. Survival that of a filterE. coli coated bioaerosols with through a higher the concentration chitosan-coated of and chitosan untreated results filters. in a greater antimicrobial effect against B. subtilis bioaerosols.

Figure 4. Survival of B. subtilis bioaerosols through the chitosan-coated and untreated filters. Figure 4. Survival of B. subtilis bioaerosols through the chitosan-coated and untreated filters.

Furthermore, these CCFs had a higher ability to inactive E. coli bioaerosols (REc = 46%, 3.3. Effect of Face Velocity on Bioaerosol Survival through CCFs 38%, and 23% with 1.0%, 1.5%, and 2.5% CCFs, respectively) than B. subtilis bioaerosols (RBs =Figure 50%, 346%, shows and the 34% survival with (R 1.0%,Ec) of 1.5%, the E. andcoli bioaerosols 2.5% CCFs, through respectively), the 1.0%, mainly 1.5%, dueand 2.5%to the CCFs fact thatat face Gram-negative velocities of 10,E. 20, coli andis a 30 bacterial cm/s (RH strain 30%), that respectively. is sensitive The to antimicro-REC of the E.bial coli agents, bioaerosols while through Gram-positive the 2.5%B. CCF subtilis increasedis a resistant from 23% bacterial to 37% strain as the [22 face,23 ]velocity with a increasedmainly peptidoglycan-consisted from 10 to 30 cm/s. As shown cell wall in structure.Figure 4, the Therefore, survival compared (RBs) of B. withsubtilisB. bioaer- subtilis osolsbioaerosols, throughE. colithe bioaerosols2.5% CCFs areincreased more antimicrobial from 34% to agent 48% sensitive as the face and velocity easily biologically increased frominactivated 10 to 30 and cm/s. removed The results from indoordisplay air that by the CCFs. REc of This E. coli finding through is consistent these three with CCFs the increasedresults of anotherwith the bioaerosol face velocity, control which approach indicates in athat previous the biological study of inactivating Lin and Li [ability6]. of the CCFs decreased with the increase in face velocity. This is probably due to the increase in3.3. face Effect velocity of Face Velocity(mechanical) on Bioaerosol reducing Survival the residence through CCFs time associated with bioaerosols Figure3 shows the survival (R Ec) of the E. coli bioaerosols through the 1.0%, 1.5%, and 2.5% CCFs at face velocities of 10, 20, and 30 cm/s (RH 30%), respectively. The REC of the E. coli bioaerosols through the 2.5% CCF increased from 23% to 37% as the face velocity increased from 10 to 30 cm/s. As shown in Figure4, the survival (R Bs) of B. subtilis bioaerosols through the 2.5% CCFs increased from 34% to 48% as the face velocity increased from 10 to 30 cm/s. The results display that the REc of E. coli through these three CCFs increased with the face velocity, which indicates that the biological inactivating ability of the CCFs decreased with the increase in face velocity. This is probably due to the increase in face velocity (mechanical) reducing the residence time associated with bioaerosols attraction (the majorly related to electrostatic-capturing force, which is weaker than mechanical force) to the chitosan on the surface of the CCFs [26,27]. According to the experimental data, the antimicrobial effect on CCFs decreased as the face velocity increased, which could be attributed to the shorter residence time.

3.4. Effect of Relative Humidity on Survival through the CCFs

Figures5 and6 show the plot of survival of E. coli (REc) and B. subtilis (RBs) bioaerosols through the 1.0%, 1.5%, and 2.5% CCFs at RH values of 30%, 50%, and 70% (under face velocity of 10 cm/s), respectively. According to these results, the survival of the two bacterial bioaerosols (REc and RBs) through CCFs increased with the RH. This is probably due to the fact that as the inactivating test was controlled under the condition of higher RH, the fiber surface of the CCFs accumulated a higher water content. Chitosan is hygroscopic and has a high capability to form hydrogen bonds. However, it might interfere with the electrostatic-capturing force (majorly generated by charged NH3+ group of chitosan) or alter the polymer’s physicochemical and biological properties [29,30]. This mechanism would decrease the biological inactivating ability of CCFs. Int. J. Environ. Res. Public Health 2021, 18, x 8 of 12

Int. J. Environ. Res. Public Health 2021, 18, x 8 of 12

attraction (the majorly related to electrostatic-capturing force, which is weaker than me- chanicalattraction force) (the majorlyto the chitosan related onto theelectrostati surface c-capturingof the CCFs force, [26,27]. which According is weaker to the than exper- me- imentalchanical data,force) the to theantimicrobial chitosan on effect the surfac on CCFe ofs thedecreased CCFs [26,27]. as the Accordingface velocity to increased,the exper- whichimental could data, be the attributed antimicrobial to the effectshorter on residence CCFs decreased time. as the face velocity increased, which could be attributed to the shorter residence time. 3.4. Effect of Relative Humidity on Survival through the CCFs

3.4. EffectFigures of Relative 5 and 6 Humidityshow the onplot Survival of survival through of E. the coli CCFs (REc ) and B. subtilis (RBs) bioaerosols

throughFigures the 1.0%,5 and 1.5%,6 show and the 2.5% plot ofCCFs survival at RH of values E. coli (RofEc 30%,) and 50%, B. subtilis and 70%(RBs) (underbioaerosols face velocitythrough ofthe 10 1.0%, cm/s), 1.5%, respectively. and 2.5% According CCFs at RH to valuesthese results, of 30%, the 50%, survival and 70% of the (under two bac-face terialvelocity bioaerosols of 10 cm/s), (REc respectively. and RBs) through According CCFs increasedto these results, with the the RH. survival of the two bac- terialThis bioaerosols is probably (REc dueand toRBs the) through fact that CCFs as the increased inactivating with test the wasRH. controlled under the conditionThis isof probably higher RH, due the to fiber the fact surface that ofas the the CCFs inactivating accumulated test was a hi controlledgher water under content. the Chitosancondition is of hygroscopic higher RH, theand fiber has surfacea high capabilityof the CCFs to accumulated form hydrogen a hi bonds.gher water However, content. it mightChitosan interfere is hygroscopic with the electrostatic-captur and has a high capabilitying force to (majorly form hydrogen generated bonds. by charged However, NH3 it+ groupmight interfereof chitosan) with or the alter electrostatic-captur the polymer’s physicochemicaling force (majorly and generated biological by properties charged NH3 [29]+ Int. J. Environ. Res. Public Health 2021,[30].group18, 7183 This of chitosan)mechanism or wouldalter the decrease polymer’s the biologicalphysicochemical inactivating and biological ability of CCFs.properties 8 of[29] 11 [30]. This mechanism would decrease the biological inactivating ability of CCFs.

Figure 5. Survival of E. coli bioaerosols through the chitosan-coated and untreated filters in different Figure 5. Survival of E. coli bioaerosols through the chitosan-coated and untreated filters in different relativeFigure 5. humidity. Survival of E. coli bioaerosols through the chitosan-coated and untreated filters in different relativerelative humidity.humidity.

Figure 6. Survival of B. subtilis bioaerosols through the chitosan-coated and untreated filters in Figure 6. Survival of B. subtilis bioaerosols through the chitosan-coated and untreated filters in dif- different relative humidity. ferentFigure relative 6. Survival humidity. of B. subtilis bioaerosols through the chitosan-coated and untreated filters in dif- 3.5.ferent Application relative humidity. of CCFs in Field Indoor Environments

This work applied CCFs in a real indoor environment to understand their bioaerosol field removal capacity. A dental clinic was chosen as the testing room. The size of the testing room was 3 × 3 × 1.8 m3. A self-made air-cleaning device was fabricated with a 220 × 200 × 160 mm3 stainless-steel body-box and DC-powered fan (12V, diameter 40 mm, mounted in air intake side). A diameter 100 mm filter holder was installed on the opposite side of the body-box as an exhaust (shown as Figure7). An untreated air filter or 1.5% CCF could be placed into the holder to clean the indoor air intake using a fan with 10 cm/s velocity. This self-made air-cleaning device was applied approximately 3 h after the end of a day-long dental operation. The bacterial bioaerosols were sampled in triplicate by an Andersen one-stage sampler at intervals of 30 min, followed by the collection and cultivation process described in Section 2.4. The background average bacteria bioaerosol concentration in this test room was monitored and calculated as 1513 ± 145 CFU/m3 before applying the air-cleaning device. Figure8 shows that the bacteria bioaerosol removal efficiency when using the 1.5% CCFs and untreated filters in the testing room. During the testing process, the average RH was 55.5%, and average temperature was 25.6 ◦C. The experimental result indicates that the bacteria bioaerosol removal efficiencies when using the 1.5% CCFs and untreated filters were 32.6% and 80.1%, respectively. This preliminary, single-shot finding implies that the CCFs have potential for indoor air cleaning. Int. J. Environ. Res. Public Health 2021, 18, x 9 of 12 Int. J. Environ. Res. Public Health 2021, 18, x 9 of 12

3.5. Application of CCFs in Field Indoor Environments 3.5. Application of CCFs in Field Indoor Environments This work applied CCFs in a real indoor environment to understand their bioaerosol field removalThis work capacity. applied A CCFs dental in aclinic real indoor was chosen environment as the testingto understand room. The their size bioaerosol of the testingfield removal room was capacity. 3 × 3 × 1.8A dentalm3. A self-made clinic was air-cleaning chosen as thedevice testing was room.fabricated The withsize aof 220 the 3 ×testing 200 × 160room mm was3 stainless-steel 3 × 3 × 1.8 m . body-boxA self-made and air-cleaning DC-powered device fan was(12V, fabricated diameter with 40 mm, a 220 3 mounted× 200 × 160in air mm intake stainless-steel side). A diameter body-box 100 mmand filterDC-p holderowered was fan installed (12V, diameter on the opposite 40 mm, sidemounted of the in body-box air intake as side). an exhaust A diameter (shown 100 mmas Figure filter holder7). An wasuntreated installed air on filter the oropposite 1.5% CCFside could of the be body-box placed into as thean exhaustholder to (shown clean th ase indoorFigure air7). intakeAn untreated using a fanair withfilter 10 or cm/s 1.5% velocity.CCF could This be self-made placed into air-cleaning the holder deviceto clean was the appliedindoor air approximately intake using a3 fanh after with the 10 endcm/s ofvelocity. a day-long This dental self-made operation. air-cleaning The bacterial device bioaerosolswas applied were approximately sampled in 3triplicate h after the by endan Andersenof a day-long one-stage dental sampler operation. at intervalsThe bacterial of 30 bioaerosols min, followed were by sampled the collection in triplicate and culti- by an vationAndersen process one-stage described sampler in Section at intervals 2.4. The of background 30 min, followed average by bact the eriacollection bioaerosol and con-culti- centrationvation process in this described test room in was Section monitored 2.4. The and background calculated average as 1513 bact ± 145eria CFU/m bioaerosol3 before con- 3 applyingcentration the in air-cleaning this test room device. was Figuremonitored 8 sh owsand thatcalculated the bacteria as 1513 bioaerosol ± 145 CFU/m removal before ef- ficiencyapplying when the air-cleaningusing the 1.5% device. CCFs Figure and untreated 8 shows thatfilters the in bacteria the testing bioaerosol room. Duringremoval the ef- testingficiency process, when usingthe average the 1.5% RH CCFs was and 55.5%, untreated and average filters intemperature the testing wasroom. 25.6 During °C. The the experimentaltesting process, result the indicates average thatRH thewas bacteria 55.5%, bioaerosoland average removal temperature efficiencies was when25.6 °C. using The Int. J. Environ. Res. Public Health 2021,the18experimental, 71831.5% CCFs resultand untreated indicates filters that the were bacteria 32.6% bioaerosoland 80.1%, removal respectively. efficiencies This preliminary, when9 using of 11 single-shotthe 1.5% CCFs finding and implies untreated that filters the CCFs were have32.6% potential and 80.1%, for respectively.indoor air cleaning. This preliminary, single-shot finding implies that the CCFs have potential for indoor air cleaning.

Figure 7. Schematic diagram of self-made air-cleaning device. FigureFigure 7. 7.Schematic Schematic diagram diagram of of self-made self-made air-cleaning air-cleaning device. device.

Figure 8. SurvivalSurvival of bacterial bioaerosols by using the 1.5% chitosan-coated and untreated filtersfilters in theFigure testing 8. Survival room. of bacterial bioaerosols by using the 1.5% chitosan-coated and untreated filters in the testing room. 3.6. Evaluating the Effects of Operating Parameters on CCFs’ Bioaerosol Removal Characteristics

To characterize the bioaerosol removal performance of CCF, this work aimed to build a regression equation to integrate various parameters’ effects on the survival of bacterial bioaerosols through CCFs. The multiple regression analysis was used to understand the effects of different parameters on bioaerosol inactivating. This model was applied in our pervious study [31]. According to the results, the survival behaviors of E. coli and B. subtilis bioaerosols were obviously different due to their specific biological characteristics (Gram-positive and -negative). Hence, two regression equations were calculated for these two bioaerosols, respec- tively. The regression equation considered the chitosan concentration (1.0%, 1.5%, and 2.5%), face velocity through the CCFs (10, 20, and 30 cm/s) and RH (30%, 50%, and 70%). The regression equation was constructed as follows.

S = aWbUcHd (1)

where a, b, c, and d are the regression constants; S is the total survival rate through the CCFs; W is the chitosan concentration (%); U is the face velocity (m/s); and H is the RH (%). According to the regression analysis, the regression equations for each E. coli and B. subtilis bioaerosols are as follows: S = 0.65W0.25U−0.14H−0.08 (2) S = 0.38W0.20U−0.11H−0.05 (3) Based on the regression analysis, the correlation coefficients R2 were approximately 0.83 and 0.81 for the regression equations for E. coli and B. subtilis bioaerosols, respectively. Int. J. Environ. Res. Public Health 2021, 18, 7183 10 of 11

By comparing the coefficients in each equation, the effects of the different parameters on bioaerosol survival were demonstrated. The regression analysis results show that in each regression equation, the constant b is largest and indicates that the “chitosan-coating concentration” has the greatest influence on the inactivating ability of the CCFs among all influencing factors. According to these regression data, “face velocity” has the second highest impact on the survival of CCFs, and the effect of the RH on survival is the lowest, based on our experimental data. In this work, we chose E. coli and B. subtilis as the Gram- negative and Gram-positive analyzed bioaerosols, which are common and reliable options in bioaerosols studies. However, there are still many microorganisms in a broader sense of biological characteristics (such as yeast, fungi, and ). The degree of influence of these three factors only existed under the controlled condition of the experiment and was limited by the chosen bioaerosols.

4. Conclusions Bioaerosols cause various adverse health effects and have a significant impact on indoor air quality. The air-cleaning technology must be effective, cost beneficial, environ- mentally friendly, and safe for humans. In this work, we attempted to enhance bioaerosol removal efficiency by means of simply coating a commercial air filter with chitosan. Within the laboratory experiments of non-biological PSL aerosol and bioaerosols (including E. coli and B. subtilis), the CCFs were proven to have additional removal capability due to their antimicrobial properties. Moreover, the installed CCF self-made air-cleaning device showed potential in removing bacterial bioaerosols in the field environment. Among the operating parameters influencing CCF removal performance, we used re- gression analysis to show that the chitosan-coating concentration has the greatest influence. The face velocity and RH, on the other hand, have mild effects on bioaerosol removal. The approaches of simply preparing a chitosan-coated filter and performance characterization we used in this work will be further improved and utilized to test various antimicrobial agents and bioaerosols.

Author Contributions: Conceptualization, S.Y.; methodology, S.Y. and Y.-F.H.; software, S.Y. and C.-Y.C.; validation, S.Y. and Y.-F.H.; formal analysis, S.Y.; resources, S.Y.; data curation, S.Y. and C.-Y.C.; writing—original draft preparation, S.Y.; writing—review and editing, S.Y. and C.-Y.C.; visualization, S.Y. and C.-Y.C. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the Nation al Science Council of Republic of China project. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data supporting the results in the current study are available from the corresponding author on reasonable request. Conflicts of Interest: The authors declare no conflict of interest.

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