PLA nonwovens modified with poly(dimethylaminoethyl methacrylate) as antimicrobial filter materials for workplaces

Abstract

The objective of the present study was to assess the antimicrobial effectiveness of silver- and copper-modified polylactide (PLA)–poly(dimethylaminoethyl methacrylate) (PDMAEMA) nonwovens used as materials for the production of air purifying respirators. The antimicrobial activity of six types of PLA nonwovens, with different PDMAEMA, Ag, and Cu contents, were tested using the ATCC 100 method. Microorganisms were isolated from the air in 12 workplaces within composting plants, tanneries, and museums. Dominant bacterial and fungal species were identified using 16S RNA and ITS1/2 molecular analysis, respectively. Air samples collected from the composting plant and tannery were highly contaminated with bacteria, while those from museums with fungi (10³–10⁴CFU/m³). We identified potentially pathogenic microorganisms from the following genera: Bacillus, Corynebacterium, Pseudomonas, Candida, Aspergillus, Penicillium, Scopulariopsis, and Paecilomyces. PLA nonwovens containing 5.1% poly(dimethylaminoethyl methacrylate) and 2.7% Ag or 3.8% Cu exhibited good antimicrobial properties (R > 99.9%) against the pathogenic strains found in the above workplaces. These nonwovens also have good inspiratory resistance parameters.

Keywords

microorganism workplaces bioactive half-mask polylactide poly(dimethylaminoethyl methacrylate)copper

Workers are often exposed to biological hazards such as bioaerosols in workplaces. According to the literature, the workplaces of composting facilities have significant microbiological hazards, including pathogenic microorganisms such as Pasteurella sp., Proteus mirabilis sp., Streptomyces sp., Corynebacterium sp., and Aspergillus fumigatus.^1–3 Similarly, the materials processed in tanneries cause extensive microbial growth. Microbes isolated from these workplaces include bacteria, which attack processed and finished leather, and those that pose a threat to workers’ health. These include Staphylococcus aureus, Bacillus anthracis, and B. subtilis.^4,5 The materials stored in museums (e.g. paper, wood, fabrics) are readily colonized by microorganisms under favorable temperature and humidity conditions. The bioaerosol of museum rooms is rich in filamentous fungi of the genera Alternaria, Aspergillus, Penicillium, Cladosporium, Mucor, and Rhizopus. They are responsible both for the biodeterioration of objects and health issues, mostly allergies, in workers.⁶

Workers can be protected against these harmful microbiological factors using air-purifying respirators, whose main function is to prevent inhalation of the microorganisms present in the air and inhibit their growth or kill them.

Materials used in the production of bioactive respirators include synthetic polymers (polyester, polypropylene, polyamide, polyacrylonitrile, polycarbonate), which are modified with biocides. The biocides most frequently employed are stable quaternary ammonium salts (QACs) and metals (e.g. silver, copper) due to their strong, broad-spectrum effects on microbes, and the fact that these agents do not exhibit any toxicity or allergenicity.^7,8 Biocides should also be characterized by stability during use and storing of materials. It is known stability of QACs with the polymers. Gutarowska and Michalski showed that the antimicrobial activity of nonwovens with sanitized (containing quaternary ammonium salts) conditions, stored for half-a-year at 20℃ and RH 45%, decreased by 18–27%.⁸ While such solutions effectively eliminate microorganisms and protect the respiratory tract, which has been proven by previous research, they are a substantial burden on the environment since these polymers are poorly biodegradable.^9,10 Thus the optimal the optimum technological solutions are those that not only exhibit antimicrobial properties without irritant or allergic effects on humans, but also biodegrade at the end of their life cycle. A material that potentially fits the above criteria is the easily biodegradable polylactic acid (PLA) with the biologically active poly(dimethylaminoethyl methacrylate) (PDMAEMA). PDMAEMA is a polycationic polymer, rich in tertiary amino groups with confirmed bactericidal properties (Figure 1).^11–13

Figure 1.

Structure of poly(dimethylaminoethyl methacrylate) (PDMAEMA).

This material inhibits bacteria via direct electrostatic contact between its positively charged fiber surfaces and the negatively charged phosphatidylamine of the bacterial cell wall.¹⁴ Furthermore, PLA-PDMAEMA copolymers exhibit good biodegradation properties, which make such materials highly desirable in the market.^15,16 However, to date PDMAEMA has not been tested against microorganisms as a filter material in air purifying respirators. Its effects on fungi, which are a widespread workplace hazard, have not been studied either. In light of the above, we formulated the following research objectives: We first evaluated microbiological contamination in composting plants, tanneries, and museums, and identified the dominant microorganisms. In the next step, we determined the antimicrobial properties of silver- and copper-modified PLA–PDMAEMA nonwovens both against collection strains and strains isolated from workplaces. The strains were selected based on their frequency of occurrence and potential pathogenicity. Furthermore, we analyzed the flow resistance of the tested materials to determine their usability in producing bioactive air purifying respirators to protect the respiratory tract of workers.

Experimental details

Materials

Composition and properties of PLA–PDMAEMA nonwoven fabrics

In our experiments, we modified PLA fibers with a linear density of 6.7 dtex and a length of 60 mm (Taiwan). PDMAEMA was prepared by radical polymerization of dimethylaminoethyl methacrylate (DMAEMA) initiated with azobisisobutyronitrile (AIBN, Merck, Germany). The molecular mass of the resulting polymer (M_n = 45,340) was determined by gel permeation chromatography (GPC). In the first step of modification, different amounts of PDMAEMA were incorporated in PLA fibers, which were subsequently used to make nonwovens by the needle punch method. In this way, samples 1, 2, and 3 were produced, which differed in the amount of PDMAEMA included in the material (1.1, 2.7, and 5.1%, respectively) (Table 1). In the next step, a layer of either silver (in the form of AgNO₃, POCH, Poland) or copper (in the form of CuSO₄, POCH, Poland) was formed on the nonwoven containing 2.7% PDMAEMA, according to the specifications given in Table 1. A larger amount of copper (21.9%) was introduced to nonwoven no. 3, containing the highest concentration of PDMAEMA, due to the stoichiometry of the complexing interactions between PDMAEMA and Cu (sample 6).

Table 1.

Composition and description of modifications of the studied PLA–PDMAEMA nonwovens

No	Nonwoven – composition	Amount of modifier (%)	Description of modification method
0	Control – PLA	–	Unmodified control nonwoven
1	PLA + PDMAEMA	1.1	PLA fibers (588 g) were wetted with ethanol in order to remove any finish chemicals. They were immersed with 1% PDMAEMA solution (6 L) for 0.5 h. The solution was drained and the fibers were lightly wrung, air dried, and weighed.
2	PLA + PDMAEMA	2.7	Fibers with 1.1% PDMAEMA (325 g) were immersed in 2% PDMAEMA solution for 0.5 h; subsequent procedures as above.
3	PLA + PDMAEMA	5.1	Fibers with 1.1% PDMAEMA (300 g) were immersed in 3.5% PDMAEMA solution for 0.5 h; subsequent procedures as above.
4	PLA + PDMAEMA + Ag	2.7 + 6.0	A solution containing 1.8 g (25 mmol) of poly(acrylic acid) (PAA, MW = 450,000, Aldrich) in 100 mL of water was prepared. Then, 25 mL 0.1 M AgNO₃ (POCH, Poland) was mixed with 25 mL 0.1 M NaOH, and the AgOH precipitate was washed with water and drained; subsequently PAA solution was added and the mixture was stirred vigorously. The solution contained PAA substituted in 10% with Ag⁺ ions. Nonwovens with 2.7% PDMAEMA were immersed in this solution for 0.5 h, and subsequently air dried and weighed.
5	PLA + PDMAEMA + Cu	2.7 + 3.8	The nonwoven with 2.7% PDMAEMA (6 g) was immersed in 200 mL of 2% CuSO₄ for 0.5 h, and then washed with distilled water; subsequent procedures as above.
6	PLA + PDMAEMA + Cu	5.1 + 21.9	The nonwoven with 5.1% PDMAEMA (55.2 g) was sprayed with 2% CuSO₄ (about 1.8 dm³); subsequent procedures as above.

For comparison, we produced a nonwoven made of unmodified PLA fibers (sample 0) with properties as close as possible to those of the nonwovens made from modified fibers. The control nonwoven had the following properties: surface density 197.9 g/m², thickness 4.21 mm, apparent density 47.0 kg/m³, air permeability at a pressure difference Δp of 100 Pa = 1621 L/m²/s. These properties met the requirements specified for preliminary filters in air purifying respirators.

Methods

Surface and apparent density measurements

Surface density and apparent density of the nonwovens were measured according to the EN 290731:1999 standard, while inspiratory resistance was measured according to EN 143:2004/A1:2007 at an air flow of 30 L/min and 95 L/min.^17,18 In both cases possible measurement error was ±5%.

EDS and SEM analysis

The surface of the fibers before and after modification was studied using a Nova Nano SEM 230 scanning electron microscope (from FEI) with an EDS Apollo 40 SDD X-ray microanalyzer from EDAX. All measurements were performed with a random error of 5%.

Thermogravimetric analysis

The thermal analysis of all samples was carried out with a PerkinElmer TGA 7 thermal analyzer in a platinum measuring cell. The Pyris program was used for data handling. The measurements were performed in a nitrogen and air atmosphere with the heating rate 15℃/min. The samples were heated up to 650℃, starting at room temperature. All measurements were repeated at least three times.

Assessment of air microbial contamination at workplaces

Microbiological contamination was analyzed at four tanneries, four composting plants, and four museums (in total: 37 workplaces). Control analysis was also undertaken in the atmospheric air at each site. The temperature and humidity of the air were determined using a PWT-401 hygrometer (Elmetron, Poland). Descriptions of the tested workplaces are given in Table 2.

Table 2.

Characteristics of the examined workplaces

Work environment	C (m³) T (℃) (SD) RH (%) (SD)	Description of workplaces
Composting plant N = 4 Production halls n = 12	C: 500–25,317 T: 14.3 (6.5) RH: 51.3 (17.7)	Production of substrate for champignons, N = 2, n = 5 (compost production for mushroom growing; raw materials used: wheat and rye straw, poultry and horse manure; production and sale of substrate with oversized mycelium in the form of cut blocks and in bulk for mushroom production); Municipal plants N = 2, n = 7 (compost production from green waste, sorting of green waste prior to composting, raw material stockpiling, forming and turning of compost heaps composting boxes, room with equipment for controlling and monitoring the processes in the containers)
Tanneries N = 4, Production halls n = 14	C: 250–27,000 T: 19.6 (4.7) RH: 49.5 (8.5)	Raw leather N = 2, n = 8 (movement and segregation of raw material, storage of salted raw hides, preliminary and main soaking, rinsing of hides, fleshing, liming, bating, pickling, cutting of wet-blue hides, storage of wet-blue and tanned hides) Wet blue hides (chrome-tanned) N = 2, n = 6 (re-tanning and finishing of wet-blue leather, short-term storage of palettes of raw material, vacuum drying of hides, movement of hides using hoists, short-term storage of hides)
Museums N = 4, Warehouses n = 14	C: 52–3055 T: 18.4 (3.7) RH: 38.7(2.9)	Museum of Textile n = 4 (collects machine: wood, steel for processing fibers, mainly cotton and linen; collection of materials on wooden and steel shelves; restoration workshop - washing, drying and preliminary textile repair) Museum of Independence Tradition n = 3 (collection of paintings on gantries, weapons: swords, rifles, pistols; collection of flags, banners gathered in wooden dressers; collection of flags, banners gathered in wooden dressers) Museum of Archeology and Ethnography n = 3 (furniture warehouse, department of folk art – collection of wooden furniture, sculptures; section of farm and rural industry – collection of materials such as wood, wicker, iron; magazine of African cultures, collection of clothing, masks, weapons, sculptures) National Museum n = 4 (collection of oil paintings on canvas and board; collection of sculptures from this period; medieval art restoration workshop)

C – cubage; T – average air temperature; RH – average air relative humidity; SD – standard deviation; N – number of studied institutions/plants; n – number of studied premises.

Microbiological contamination of the air was determined using an MAS-100 Eco Air Sampler (Merck, Germany). Samples of 50 and 100 liters of air were taken on DG18 agar (dichloran glycerol selective medium, Merck, Germany) medium and MEA (malt extract agar, Merck, Germany), with chloramphenicol, (0.1%) to determine total fungal number (including hydrophilic and xerophilic fungi), and on TSA medium (tryptic soy agar, Merck, Germany) with nystatin (0.2%) for total bacterial number. Air samples were taken in 6–8 repetitions for each place. Next, the samples were incubated at 30 ± 2℃ for 48 h (bacteria) and at 27 ± 2℃ for 5–7 days (fungi). Following incubation the colonies were counted, and the results were expressed in cfu/m³ air. The final result was calculated as the arithmetic mean of all repetitions.

Identification of microorganisms isolated in workplace environments

Pure cultures of bacteria and yeast were characterized macroscopically: size, shape, colony color, colony structure, color of pigment released into the culture medium; and microscopically: shape of cells, Gram-staining. Following this selected diagnostic tests of bacteria were undertaken: catalase test, oxidase test (Microbiologie Bactident Oxydase, Merck, Germany). Next, for bacteria whose frequency of occurrence was greater than 20%, API tests were performed (BioMérieux, France): API 50 CH, API STAPH, and API 20 NE. For yeasts, diagnosis was performed using the API C AUX test. All filamentous fungi were identified using macroscopic and microscopic observations of strains cultured on CYA medium (Difco, USA) and YES (yeast extract with supplements), using taxonomic keys.

Strains of microorganisms most frequently present in workplaces and those with potentially pathogenic properties were used for testing antimicrobial activity of PDMAEMA nonwovens. Bacteria were identified using nucleotide sequence analysis of the 16S rRNA gene.¹⁹ Yeast and filamentous fungi were identified using the ITS1/2 sequence of the rDNA region.²⁰ The sequences obtained were deposited in the National Center for Biotechnology Information GenBank database.

Microorganisms

Antimicrobial activity of modified PDMAEMA was tested against five strains from pure culture collections, including four from ATCC (American Type Culture Collection): Escherichia coli ATCC 10536, Staphylococcus aureus ATCC 6538, Candida albicans ATCC 10231, Aspergillus niger ATCC 16404; and one from National Collection of Agricultural and Industrial Microorganisms: Bacillus subtilis NCAIM 01644. In addition, the antimicrobial activity was determined against six strains isolated from working environments, including two from composting plants: Aspergillus fumigatus KC456184, Penicillium crustosum KC456186; two from tanneries: Bacillus subtilis KC182059, Candida parapsilosis KC182053; and two from museums: Staphylococcus haemolyticus KC182061, Aspergillus versicolor KC456175.

A total of 11 microorganisms were tested. ATCC strains were selected based on the taxonomic variety and differing sensitivities to biocides. Environmental strains were selected based on their phylogenetic relationships with respective genera and strains from pure culture collections (ATCC), their frequency of occurrence in the air and harmfulness to health according to Directive 2000/54/EC, classification BSL (biosafety levels based on European Confederation of Medical Mycology (ECMM)).

Estimation of the antimicrobial activity of PDMAEMA nonwovens

The antimicrobial activity of nonwovens was measured using a modified quantity method, AATCC 100.¹⁰ Modifications: time of incubation following specifications for protective half-mask usage – 8 h is the maximum time. The percentage reduction in microorganisms after 8 h (R) was calculated according to the equation:

R = \frac{A - B}{A} \times 100 %

(1)

where A is the number of microorganisms in the control sample at time t = 0 and B is the number of microorganisms in the bioactive nonwoven after time t = 8 h.

An evaluation scale of the antimicrobial activity of PDMAEMA nonwovens was established: R ≥ 99% – high antimicrobial activity; R = 90–98% – average antimicrobial activity; R = 50–89% – low antimicrobial activity; R < 50 – lack of antimicrobial activity.

Mathematical and statistical analysis

The frequency of occurrence of a particular species at a given workplace (ƒ) was calculated according to the formula:

f = \frac{a}{n} \times 100 %

(2)

where a is the number of samples in which the strain occurred and n is the total number of samples.

Differences between the number of microorganisms in the bioactive nonwovens and control samples were analyzed using one-way analysis of variance (ANOVA). Differences were considered significant at p < 0.05. All data were analyzed using Statistica 6.0 software (Statsoft, USA).

Results

Quantitative analysis of microbiological air contamination showed a high number of airborne microorganisms in the workplaces investigated. The highest concentration of fungi was observed in museums, with a mean of 3.6 × 10³CFU/m³. Fungal contamination in composting plants and tanneries was also high and amounted to 2.2 × 10³CFU/m³ and 2.4 × 10³CFU/m³, respectively. Composting plants had the highest bacterial count in the air amongst the workplaces studied (1.0 × 10⁴CFU/m³), followed by tanneries (1.7 × 10³CFU/m³) and museums (4.2 × 10²CFU/m³) (Table 3).

Table 3.

Analysis of microbial contamination at the workplaces

Work environment	Number of microorganisms in the air (cfu/m³)		Prevailing microorganisms f ≥ 50% (max. frequency of isolation %)	Pathogens* f ≥ 20% (max. frequency of isolation %)
Work environment	Fungi	Bacteria		Pathogens* f ≥ 20% (max. frequency of isolation %)
Composting plants	M: 2.16 × 10³ Min: 3.30 × 10² Max: 1.40 × 10³ SD: 1.43 × 10³	M: 9.98 × 10³ Min: 1.10 × 10³ Max: 2.10 × 10⁴ SD: 8.71 × 10⁴	Bacteria: Bacillus sp. (80), B.megaterium (50), Brevibacterium sp. (80), Geobacillus sp. (89), G.stearothermophilus (100), G.thermoglucosidasius (61), K.varians (80), Kytococcus sedentarius (90), Leuconostoc mesenteroides (56), Methylobacterium mesophilicum (78), Micrococcus lylae (100), Staphylococcus epidermidis (100), S.lentus (61), S.xylosus (70) Fungi: Alternaria chartarum (64), Cladosporium cladosporioides (79), C.macrocarpum (86), Kloeckera sp. (86), Rhodotorula glutinis (50), Trichoderma koningii (50)	Bacteria: Bacillus cereus (56), B.subtilis (40) Pseudomonas stutzeri (78), Erysipelothix rhusiopathiae (25) Fungi: Aspergillus fumigatus (27), A.niger (79), A. flavus (64), Candida albicans (20), Cladosporium herbarum (56), Fusarium solani (33), Scopulariopsis brevicaulis (33), Mucor pirimiformis (71), M. racemosus (50), Penicillium carneum (90), P.coprobium (80), P.expansum (22), P.freii (88), P.glabrum (40), P.implicatum (40), P.lividum (22), P.crustosum (100), P.spinulosum (88)
Tanneries	M: 2.40 × 10³ Min:3.10 × 10² Max: 7.30 × 10³ SD: 2.86 × 10³	M: 1.68 × 10³ Min:5.10 × 10² Max:3.30 × 10³ SD: 1.18 × 10³	Bacteria: B.mycoides (79), Brevundimonas vesicularis (83), Kocuria rosea (79), K. varians (78), Micrococcus sp. (81), M.lylae (100), M.luteus (93), Staphylococcus cohnii (71), S.lentus (86), S.hominis (95) Fungi: Candida parapsilosis (62), Cladosporium cladosporioides (79), C.macrocarpum (78), Cryptococcus albidus (78), C.terreus (83), Rhodotorula glutinis (72)	Bacteria: Corynebacterium accolens (100), C.propionicum (79), Bacillus subtilis (27), Pseudomonas alcaligenes (33), P.fluorescens (57) Fungi: Alternaria alternata (39), A.versicolor (25), Cladosporium herbarum (38), Fusarium oxysporum (29), Mucor plumbeus (22), Paecilomyces variotii (35), Penicillium atramentosum (92), P.chrysogenum (64), P.griseofulvum (73), P.hirsutum (54), P.lividum (88), P.oxalicum (36), P. palitans (33), P.polonicum (46), P.verrucosum (86)
Museums	M: 3.64 × 10³ Min: 7.50 × 10¹ Max: 1.40 × 10⁴ SD: 5.98 × 10⁴	M: 4.15 × 10² Min: 3.40 × 10² Max: 5.60 × 10² SD: 8.98 × 10¹	Bacteria: Bacillus firmus (75), Chryseobacterium indologenes (88), Kocuria kristinae (83), K.varians (75), Micrococcus sp. (100), Staphylococcus haemolyticus (100), S.lentus (100), S.hominis (92), S.xylosus (75) Fungi: Aspergillus sp. (100), C. macrocarpum (50), Rhizopus nigricans (100)	Bacteria: Pseudomonas alcaligenes (75) Streptomyces sp. (50) Fungi: Aspergillus niger (21), A.versicolor (61), Cladosporium herbarum (39), Mucor sp. (83), Penicillium carneum (46), P.commune (33), P. paneum (25), P.radicola (46)

M – arithmetic mean; Min/Max – minimum/maximum value; SD – standard deviation; nt - not tested; (-) – not detected; * – according to Directive 2000/54/EC, classification BSL (Biosafety levels based on European Confederation of Medical Mycology (ECMM).

Strains used for the tests of antimicrobial activity of PDMAEMA nonvowens are underlined.

The most frequently (≥90%) isolated bacteria in composting plants were: Geobacillus sp., G. stearothermophilus, Kytococcus sedentarius, Micrococcus lylae, and Staphylococcus epidermidis, while in tanneries M. lylae, M. luteus, and Staphylococcus hominis were the most frequent. The microorganisms most widespread in museums were the bacteria Micrococcus sp., Staphylococcus haemolyticus, S. lentus, S. hominis, and the fungi Aspergillus sp., Rhizopus nigricans (Table 3).

A number of bacterial and fungal species isolated from the workplaces investigated, were potentially hazardous to workers’ health. These include Pseudomonas stutzeri, Bacillus subtilis, Corynebacterium propionicum, Candida albicans, Aspergillus fumigatus, Scopulariopsis brevicaulis, Paeciliomyces variotii, and many species from the genus Penicillium (Table 3).

The physical and flow properties of the studied bioactive nonwovens are given in Table 4.

Table 4.

Physical and flow properties of the studied PLA-PDMAEMA nonwovens

Sample	Type and amount of modifier^a	Mass per unit area (g/m²)	Apparent density (kg/m³)	Breathing resistance with 30 L/min, (mbar)	Breathing resistance with 95 L/min, (mbar)
0	Unmodified	197.9	47.0	0.0134	0.0118
1	PDMAEMA – 1.1 %	224.2	55.9	0.0021	0.0423
2	PDMAEMA – 2.7 %	195.7	44.8	0.0068	0.0033
3	PDMAEMA – 5.1 %	190.7	60.3	0.0221	0.0275
4	PDMAEMA + Ag – 2.7 % + 6.0%	206.7	63.19	–	–
5	PDMAEMA + Cu – 2.7% + 3.8%	200.0	53.7	0.0085	0.0029
6	PDMAEMA + Cu – 5.1% + 21.9%	211.2	71.12	0.0285	0.0127

The amount of modifier was additionally confirmed by dissolving of PDMAEMA and acidic titration of the substance for samples 1–3, or by thermogravimetric analysis (increase in solid residue) for samples 4–6. Calculated error in the amount of modifier is about 6%.

The structure of the bioactive nonwovens was confirmed using energy-dispersive X-ray spectroscopy (EDS). Table 5 presents EDS analysis results of the surface composition of fibers. This data confirms that the chemical composition of the fiber surface depends on the type of treatment applied. At a PDMAEMA concentration of 1.1% no nitrogen was observed on the surface. This element appears at PDMAEMA concentrations of 2.7% and 5.1%. The absence of nitrogen in sample 1 may be caused by non-uniform distribution of material on the surface, which is essential for correct detection in the EDS technique. In the other cases (2 and 3), the concentration of nitrogen on the surface was sufficient for correctly detecting X-radiation line N Ka. The formation of silver- and copper-PDMAEMA complexes also led to a change in the chemical composition of the surface (we found signals coming from silver and copper). The results presented in Table 4 suggest an increase in mass per unit area and density of the samples, following treatment with metal salts. However, this increase did not cause a dramatic change in breathing resistance measured at an airflow of 30 L/min and 95 L/min, which remained much below the permissible limit for P1 filtration materials (0.6 mbar and 2.1 mbar, respectively according to regulations).¹⁸

Table 5.

Percentage share of the studied elements on the surface of the fibers

Sample	Type and amount of modifier	Percentage share of the elements on the surface (%)
Sample	Type and amount of modifier	C	0	N	Ag	S	Cu
0	Unmodified	49.7	50.3	not observed	not observed	not observed	not observed
1	PDMAEMA – 1.1 %	46.4	53.6	not observed	not observed	not observed	not observed
2	PDMAEMA – 2.7 %	46.3	45.7	8.0	not observed	not observed	not observed
3	PDMAEMA – 5.1 %	40.2	46.0	13.8	not observed	not observed	not observed
4	PDMAEMA + Ag – 2.7 % + 6.0%	46.2	47.5	not observed	6.3	not observed	not observed
5	PDMAEMA + Cu – 2.7% + 3.8%	39.9	42.5	not observed	not observed	3.6	14.0
6	PDMAEMA + Cu – 5.1% + 21.9%	40.9	38.5	not observed	not observed	3.9	16.7

Experiments on PLA-PDMAEMA polymeric materials and their metal-modified versions (Cu and Ag), showed that these nonwovens had antimicrobial properties (Table 6). The control PLA fibers exhibited weak activity (p < 0.05) only against three strains: S. haemolyticus KC 182061, A. niger ATCC 16404, and A. versicolor KC 456175. The addition of PDMAEMA (at concentrations of 1.1%–5.1%) to PLA increased the antimicrobial activity of the nonwoven against all studied microorganisms. Even as little as 1.1% PDMAEMA completely inhibited the growth of B. subtilis KC 182059 (from tanneries) and was moderately active (R = 95.4%–98.7%) against the yeasts C. albicans ATCC 10231 and C. parapsilosis KC 182053 (from tanneries), and also against the mold A. niger ATCC 16404 (p < 0.05).

Table 6.

The antimicrobial activity of PDMAEMA nonwovens

Nonvowen (time of incubation)	Number of microorganisms (cfu/sample), Reduction (%)
	E. coli ATCC 10536	S. aureus ATCC 6538	S. haemolyticus KC182061	B. subtilis NCAIM 01644	B. subtilis KC182059	C. albicans ATCC 10231	C. parapsilosis KC182053	A. niger ATCC 16404	A. fumigatus KC456184	A. versicolor KC456175	P. crustosum KC456186
PLA (t = 0 h)	M:9.8 × 10⁶ SD:6.4 × 10⁶	M:2.9 × 10⁶ SD:4.7 × 10⁵	M:1.2 × 10⁷ SD:4.3 × 10⁶	M:7.5 × 10⁵ SD:2.6 × 10⁵	M:1.7 × 10⁶ SD:8.6 × 10⁵	M:2.6 × 10⁴ SD:8.5 × 10⁴	M:8.7 × 10⁴ SD:2.1 × 10⁴	M:1.3 × 10⁵ SD:6.8 × 10⁴	M:9.8 × 10⁴ SD:5.4 × 10⁴	M:5.6 × 10⁴ SD:1.9 × 10⁴	M:7.4 × 10⁴ SD:2.6 × 10⁴
PLA (t = 8 h)	M:3.3 × 10⁷# SD:1.8 × 10⁷ R:–	M:1.8 × 10⁷# SD:3.4 × 10⁶ R:–	M:1.4 × 10⁶# SD:1.2 × 10⁶ R:88.1	M:6.9 × 10⁶# SD:2.5 × 10⁶ R:–	M:2.5 × 10⁶ SD:1.6 × 10⁶ R:–	M:8.1 × 10⁵# SD:4.0 × 10⁵ R:–	M:9.0 × 10⁵# SD:3.5 × 10⁵ R:–	M:3.0 × 10⁴# SD:1.9 × 10³ R:77.5	M:1.4 × 10⁵ SD:8.7 × 10⁴ R:–	M:1.8 × 10⁴# SD:9.9 × 10³ R:68.4	M:4.5 × 10⁴# SD:2.1 × 10⁴ R:39.9
PLA + PDMAEMA 1.1% (t = 8 h)	M:5.8 × 10⁶ SD:2.8 × 10⁶ R:40.4	M:1.9 × 10⁶# SD:3.4 × 10⁵ R:33.4	M:2.2 × 10⁶# SD:3.3 × 10⁵ R:82.0	M:2.0 × 10⁶ SD:3.6 × 10⁶ R:–	M:0.0# SD:0.0 R:100.0	M:1.2 × 10⁴# SD:4.3 × 10³ R:95.4	M:1.1 × 10³# SD:5.6 × 10² R:98.7	M:9.2 × 10³# SD:7.3 × 10³ R:93.0	M:2.9 × 10⁴# SD:8.4 × 10³ R:–	M:1.6 × 10⁴# SD:6.0 × 10³ R:71.2	M:4.1 × 10⁴# SD:1.0 × 10⁴ R:44.4
PLA + PDMAEMA 2.7% (t = 8 h)	M:1.8 × 10⁶# SD:1.2 × 10⁶ R:81.5	M:1.3 × 10⁶# SD:2.9 × 10⁵ R:54.9	M:1.5 × 10⁵# SD:6.1 × 10³ R:98.8	M:3.5 × 10⁵# SD:2.1 × 10⁵ R:53.7	M:1.1 × 10⁴# SD:9.8 × 10³ R:99.3	M:1.3 × 10³# SD:3.6 × 10² R:99.5	M:6.1 × 10¹# SD:1.3 × 10¹ R:99.9	M:3.5 × 10³# SD:1.0 × 10³ R:97.4	M:1.8 × 10⁴# SD:4.4 × 10³ R:81.9	M:2.7 × 10⁴# SD:6.9 × 10³ R:51.4	M:2.4 × 10⁴# SD:8.1 × 10³ R:66.9
PLA + PDMAEMA 5.1% (t = 8 h)	M:1.0 × 10⁴# SD:7.9 × 10³ R:99.9	M:1.4 × 10⁴# SD:5.7 × 10³ R: 99.5	M:3.5 × 10²# SD:2.1 × 10² R:99.9	M:2.7 × 10²# SD:8.5 × 10¹ R:99.9	M:1.8 × 10²# SD:7.5 × 10¹ R:99.9	M:2.7 × 10³# SD:8.5 × 10² R:99.0	M:5.0 × 10¹# SD:1.6 × 10¹ R:99.9	M:5.2 × 10⁴# SD:1.5 × 10⁴ R:60.7	M:5.4 × 10⁴# SD:2.8 × 10⁴ R:45.0	M:4.0 × 10³# SD:8.9 × 10² R:92.8	M:2.1 × 10⁴# SD:4.6 × 10³ R:71.5
PLA + PDMAEMA 2.7% + Ag 6% (t = 8 h)	M:1.4 × 10⁵# SD:2.3 × 10⁵ R:98.5	M:5.6 × 10⁴# SD:2.2 × 10⁴ R:98.1	M:6.4 × 10⁶ SD:9.3 × 10⁶ R:47.8	M:2.3 × 10¹# SD:8.0 × 10⁰ R:99.9	M:9.0 × 10²# SD:3.6 × 10² R:99.9	M:3.4 × 10⁴# SD:9.0 × 10³ R:86.7	M:3.0 × 10⁴# SD:5.7 × 10⁴ R:65.8	M:0.0# SD:0.0 R:100.0	M:1.1 × 10²# SD:1.0 × 10² R:99.9	M:1.9 × 10²# SD:7.2 × 10¹ R:99.7	M:2.2 × 10⁴# SD:6.0 × 10⁴ R:70.5
PLA + PDMAEMA 2.7% + Cu 3.8% (t = 8 h)	M:0.0# SD:0.0 R:100.0	M:0.0# SD:0.0 R:100.0	M:0.0# SD:0.0 R:100.0	M:1.2 × 10²# SD:1.4 × 10¹ R:99.9	M:9.4 × 10⁰# SD:6.4 × 10⁰ R:99.9	M:0.0# SD:0.0 R:100.0	M:0.0# SD:0.0 R:100.0	M:2.0 × 10³# SD:6.4 × 10² R:98.5	M:1.5 × 10⁴# SD:3.6 × 10³ R:84.4	M:1.6 × 10⁴# SD:5.9 × 10³ R:71.7	M:2.4 × 10⁴# SD:6.7 × 10³ R:67.1
PLA + PDMAEMA 5.1% + Cu 21.9% (t = 8 h)	M:1.5 × 10⁴# SD:6.3 × 10³ R:99.8	M:8.3 × 10¹# SD:2.6 × 10¹ R: 99.9	M:2.2 × 10⁶# SD:8.8 × 10⁻¹ R:81.8	M:2.4 × 10⁰# SD:3.2 × 10⁰ R:99.9	M:2.7 × 10¹# SD:1.2 × 10¹ R:99.9	M:4.6 × 10¹# SD:2.0 × 10¹ R:99.9	M:0.0# SD:0.0 R:100.0	M:5.8 × 10⁴# SD:7.1 × 10³ R:56.0	M:1.6 × 10⁴# SD:6.4 × 10³ R:83.3	M:6.8 × 10³# SD:2.0 × 10³ R:87.9	M:2.0 × 10⁴# SD:5.8 × 10³ R:73.6
One-way analysis of variance (p < 0.05) results	SS 8.23 × 10¹⁵ df 7 MS 1.18 × 10¹⁵ F 28.65 p < 0.001	SS 2.39 × 10¹⁵ df 7 MS 3.41 × 10¹⁴ F 229.86 p < 0.001	SS1.21 × 10¹⁵ df 7 MS 1.73 × 10¹⁴ F 13.01 p < 0.001	SS 3.55 × 10¹⁴ df 7 MS 5.08 × 10¹³ F 21.02 p < 0.001	SS 6.16 × 10¹³ df 7 MS 8.80 × 10¹² F 22.23 p < 0.001	SS 4.99 × 10¹² df 7 MS 7.14 × 10¹¹ F 38.86 p < 0.001	SS 6.15 × 10¹² df 7 MS 8.79 × 10¹¹ F 49.87 p < 0.001	SS 1.27 × 10¹¹ df 7 MS 1.82 × 10¹⁰ F 29.04 p < 0.001	SS 1.49 × 10¹¹ df 7 MS 2.12 × 10¹⁰ F 14.98 p < 0.001	SS 1.96 × 10¹⁰ df 7 MS 2.80 × 10⁹ F 37.96 p < 0.001	SS 2.22 × 10¹⁰ df 7 MS 3.17 × 10⁹ F 17.97 p < 0.001

M – arithmetic mean; SD – standard deviation; R – reduction; – lack of the reduction; SS – sum of squares; df – degrees of freedom; MS – mean of square;

# – significantly different from control sample (one-way ANOVA, p < 0.05).

▪ – high antimicrobial activity (R ≥ 99%); ▪ – average antimicrobial activity (R = 90–98%); ▪ – low antimicrobial activity (R = 50–89%); □ – lack of antimicrobial activity (R < 50)

SEM micrographs (Figure 2) confirm that fiber modification caused structural changes in its surface. Furthermore, increasing the concentration of the applied material results in the development of conglomerates and microstructures that connect the fibers of the non-woven. The chemical-morphological analysis conducted demonstrated the formation of an outer layer in the modified material.

Figure 2.

SEM micrographs of PLA nonwoven surfaces: (a) unmodified (0), (b) with PDMAEMA layer (2.7%) (2), (c) with PDMAEMA-Ag complex (4), (d) with PDMAEMA-Cu (21.9%) complex (6).

We found that higher concentrations of PDMAEMA increased the biological activity of the nonwovens. The nonwoven with the greatest content of PDMAEMA (5.1%), had the strongest effect on all bacteria and yeasts, both on the ATCC collection strains and those isolated from workplaces (99.9% reduction at p < 0.05). The activity of 5.1% PLA–PDMAEMA nonwovens against molds was lower and amounted to between 45% and 92.8%, depending on the species.

Experiments showed that the addition of silver to the PLA–PDMAEMA fibers considerably increased their anti-mold activity (R = 70.5%–100% at p < 0.05), considerably inhibiting the growth of three out of the four studied species. Analysis of the degree of microorganism reduction, compared to the control sample, showed that Ag in the nonwoven had a much smaller inhibitory effect on bacteria and yeast growth (R = 47.8%–99.9%) compared to that of molds. In the case of the strain S. haemolyticus, no statistically significant differences were found in the number of bacteria between the sample with PDMAEMA and Ag, and the control sample (p > 0.05). This is probably due to the sensitivity of the strain to storage conditions, particularly the lack of a carbon source within the PLA nonwovens. In turn, the PLA–PDMAEMA nonwoven containing 3.8% Cu was extremely effective against bacteria and yeasts (R = 99.9%–100%), and moderately effective against molds (R = 67.1%–98.5%).

Analysis of the antimicrobial activity of modified PLA–PDMAEMA nonwovens showed that they effectively inhibited the growth of strains from workplaces. Furthermore, a comparison of their effects on the various groups of microorganisms studied showed that the moulds isolated from workplaces, A. fumigatus, A. versicolor, and P. crustosum, were more resistant to the bioactive nonwovens than the collection strain A. niger. In the case of bacteria, a similar result was obtained for the strain S. haemolyticus, isolated from a museum, as compared to S. aureus ATCC. Nonetheless, appropriate modification of nonwovens (a higher concentration of PDMAEMA and the presence of Ag) may significantly increase the effectiveness of microorganism elimination from environment.

Discussion

The workplaces studied were highly contaminated with bacteria and fungi. The number of microorganisms, especially of fungi, exceeded the permissible values (4.5 × 10²CFU/m³) proposed by the Commission of the European Communities in the report ‘Indoor Air Quality and its Impact on Man’ (1993).

The number of microorganisms found in composting plants was 9.98 × 10³CFU/m³ for bacteria and 2.16 × 10³CFU/m³ for fungi. However, some authors have reported even higher concentrations of microorganisms in such facilities, amounting to 10⁸CFU/m³ for bacteria and 10⁷CFU/m³ for fungi.²¹ It should be noted that 2 out of 4 facilities specialized in the production of substrate for the commercial cultivation of mushrooms, and thus conducted regular washing and disinfection. This may have decreased the number of microorganisms compared to green waste composting facilities, which were not disinfected. Previous studies examining air samples from composting plants have reported the presence of the following bacterial genera: Corynebacterium, Pasteurella, Proteus, Pseudomonas, Streptomyces; and the fungus Aspergillus fumigatus.^2,3 This study has also shown the presence of the bacterial genera Bacillus, Brevibacillus, Geobacillus, Kocuria, Micrococcus, and Staphylococcus and the fungi Mucor, Penicillium, and Rhizopus.

The concentration of bacterial bioaerosol at museum workplaces ranged from 3.4 × 10² to 5.6 × 10²CFU/m³, which confirmed previous studies on museums in different countries.^22–24 Literature data on quantitative analysis of microbiological contamination in museums vary greatly, from 5 × 10¹ to 3 × 10³CFU/m³. This variation is caused by many parameters, such as climate, type of building, and characteristics of the museum collection. Among workplaces studied museums had the highest concentration of molds: up to 1.4 × 10⁴CFU/m³. This figure is much higher than the contamination levels reported in literature to date, which range from 1.0 × 10⁰ to 3.5 × 10³CFU/m³. However, it should be emphasized that published studies report mycological contamination in the exhibition areas of museums, which are very different from storage areas, as the latter are less frequently ventilated or dusted. The presence of bacteria of the genera: Micrococcus, Staphylococcus, Bacillus, and Kocuria; and the fungi: Aspergillus, Cladosporium, and Rhizopus, isolated from the air of the examined museums confirm previous studies conducted in such buildings.^24–26

High numbers of microorganisms were also found in production halls and storage areas of tanneries, amounting to 2.4 × 10³CFU/m³ and 1.7 × 10³CFU/m³ for fungi and bacteria, respectively. No similar research on microbiological air analysis in this type of facility exists in published literature. The main species of airborne bacteria detected in tanneries were M. lylae, M. luteus, and S. hominis.

We determined whether the microorganisms isolated from workplaces constitute a health hazard to workers, using information from published studies.^27,28 In all examined workplaces (composting plants, tanneries, and museums), we identified species that may have allergic, immunotoxic, toxic, or infectious effects.

We showed that using bioactive PLA-PDMAEMA nonwovens, for the respiratory protection of workers, can reduce workplace microbiological hazards. Our study revealed that the growth of microorganisms frequently (61%–100%) found at workplaces (A. versicolor, C. parapsilosis, S. haemolyticus, P. crustosum) and those classified as group II health hazard (A. fumigatus, B. subtilis) can be effectively inhibited on the surface of PLA–PDMAEMA nonwovens modified with silver and copper.

Previous studies on PDMAEMA modified fibers have proven their antibacterial activity,¹³ while the present work confirmed this property against a wider spectrum of bacteria, both Gram-negative (E. coli) and Gram-positive (S. aureus, S. haemolyticus, B. subtilis), as well as against fungi, at higher PDMAEMA concentrations (yeasts C. albicans, C. parapsilosis, and molds A. niger, A. fumigatus, A. versicolor, P. crustosum). We observed high activity (p < 0.05) of PLA nonwovens with 2.7%–5.1% PDMAEMA against yeasts (a reduction of 99.5%–99.9%) and moderate activity (p < 0.05) against molds (R = 45%–97.4%).

We showed, for the first time, that biodegradable PLA–PDMAEMA polymer compositions complexed with silver and copper may be used to produce effective bioactive nonwovens with high antibacterial and antifungal properties. It was found that modification of PLA–PDMAEMA fibers with silver significantly increases their antifungal activity (R = 99.7%–100%). Silver is well known for its ability to inhibit bacterial and fungal growth. Over the years silver modification of non-biodegradable polymer nonwovens (containing, polypropylene, polyester, acrylic fiber, methyl methacrylate), has been the subject of numerous studies.^9,29–32 Some of these also concern their application for the protection of the respiratory tract.^8,10

This study has confirmed the high antimicrobial and, in particular, antifungal activity of nonwovens containing silver, and suggested their potential use in filtration systems designed for the respiratory protection of workers exposed to filamentous fungi (e.g. in tanneries and museums). Both silver-modified non-biodegradable and biodegradable nonwovens have antimicrobial activity, and this activity is mainly dependent on the concentration of the antimicrobial agents.^9,32

Furthermore, PLA–PDMAEMA nonwovens modified with copper were found to be highly active against yeasts and bacteria (R = 99.8%–100%), compared to control non-degradable nonwovens, which support previous studies on the effects of copper nanoparticles on E. coli and S. aureus as well as the highly resistant B. subtilis.³² Ruparelia et al. suggested that simultaneous addition of silver and copper to polymers may result in a synergistic effect.³³ This idea may be used in the future to design a PLA–PDMAEMA filtration nonwoven modified with both Ag and Cu, which would have a broad-spectrum of antimicrobial activity.

The formation of PDMAEMA-Cu and PDMAEMA-Ag chelated complexes is a known phenomenon. These complexes can have both inter- or intra-molecular structures (Figure 3(a) and (b), respectively). The additional advantage of creating such complexes is the resulting insoluble deposited layers. The stability of the modified polymer should be confirmed in further studies as well as the conditions for its biodegradation after an operating time.

Figure 3.

Structure PDMAEMA–copper inter and intra-molecular complexes.

The inspiratory resistance parameters of the studied PLA–PDMAEMA nonwovens and their versions modified with Cu and Ag, meet the requirements for the P1 filter class defined in standards.¹⁸ Our results show that the nonwovens may be used as a bioactive layer in the production of filtration systems in air purifying respirators, as the finished product does not cause any breathing discomfort.

Comparative analysis of collection strains and those isolated from workplaces showed that it is necessary to examine the antimicrobial sensitivity of the latter. Strains isolated from the environment had different sensitivities to modified PLA–PDMAEMA nonwovens compared to ATCC strains. Molds isolated from workplaces were more resistant than collection ones. In contrast, Gram-positive bacteria showed varying results, depending on the bacterial species and the nonwoven type used. We found that sensitivity to bioactive nonwovens depended both on the species and the place from which it was isolated. Therefore, analysis of the effectiveness of nonwovens used for personal protection equipment against biological agents should be conducted not only on collection strains, as it is required by the relevant standards, but also on strains isolated from the environment.^34,35

Conclusions

Workers’ health at composting plants, tanneries, and museums is at risk due to exposure to large numbers of airborne microorganisms. Polylactide material containing PDMAEMA modified with silver or copper efficiently inhibits the growth of both collection strains and potentially pathogenic ones often isolated from workplaces. Thanks to their desirable properties – antimicrobial effects and low inspiratory resistance – PLA–PDMAEMA/Ag and PLA–PDMAEMA/Cu nonwovens can be employed as filtration materials in air purifying respirators used at workplaces with microbiological hazards. Finally, while evaluating nonwovens for the protection of workers against harmful microbiological agents, their antimicrobial activity should be tested against microorganisms isolated from workplaces.

Footnotes

Funding

This work was supported by the European Regional Development Fund (grant number POIG.01.03.01-00-007/08-00) within the framework of the key project titled ‘Biodegradable fibrous products’ (acronym: Biogratex).

Acknowledgments

Studies in composting plants and tanneries were realized within the project of the Polish National Center for Research and Development, no. III.B.03 titled ‘Development of principles for the evaluation and prevention of hazards caused by biological agents in the working environment using indicators of microbial contamination’. We would like to express our gratitude to Michał Puchalski, PhD for analyzing the surface composition of modified nonwovens using the EDS and SEM methods.

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