Buffering and Antibacterial Properties of Cotton Canvas with Dolomite/ZnO-Styrene-Acrylic Complex Coating and their Comparison with Properties after the Accelerated Aging

In this study, we report evaluation of buff ering and self-sterilizing coating complex obtained by dolomite and zinc oxide particles incorporation into styrene–acrylic layer applied to cotton fabric. Surface properties of the coating were evaluated by SEM, EDS and 3D optical microscopy. Antimicrobial properties were determined using a mixture of G+ and Gbacteria (Escherichia coli, Staphylococcus epidermidis and Streptococcus mutans) that was in dynamic contact with canvases for 4-100 hours. Alkalizing buff er capacity of the surface layer supplied to the system by dolomite was tested by the addition of acetic acid. All these properties were simultaneously tested on the same canvases that have been previously exposed to the so-called hot (105 °C) and wet aging (80 °C, 65% relative humidity). The aging was provided in the climatic test room for 144 hours and the properties of canvases before and after aging were compared and evaluated.


Introduction
Composite materials based on metal (nano)particles attract signi cant attention due a wide range of physical-chemical properties. ese materials are not simply physical mixtures and they can be de ned as complex materials with organic and inorganic components intimately mixed. Modi cation of coating polymeric matrices leads to novel properties such as growth prevention or adhesion reduction of microorganisms.
ere is a signi cant interest in the development of antimicrobial and durable materials for application in the food and biomedical industries. ese materials are also used in the elds, such as archiving, museums, librarianship, preservation or storage, where extension of lifetime of the books and other ageing objects is a necessity. ese materials should combine desirable attributes such as potent bactericidal and fungicidal e ciency, environmental safety, low toxicity, and easy, cost-e ective fabrication. Carbonates are salts of carbonic acid and occur in the form of minerals and rocks commonly found in nature. Especially alkaline earth metal carbonates (Ca 2+ and Mg 2+ ) are very widely used in many productions and industrial processes in various industries like construction [1], food [2], metallurgy [3], treatment of contaminated water and soil [4,5], and fertilizers [6]. Calcium carbonate CaCO 3 (limestone) occurs in many crystalline modi cations (calcite, aragonite, alabaster, travertine, marble). Also, magnesium carbonate MgCO 3 and calcium magnesium carbonate CaMg(CO 3 ) 2 are naturally found in many modi cations, varieties and with di erent chemical impurities (Fe, Mn, Zn, Pb and others) [7]. Dolomite is a non-toxic, inexpensive, widely available double carbonate with slightly alkaline and buering capabilities. It crystallizes in the trigonalrhombohedral system and depending on the admixture it is white, gray or pink. Unlike bicarbonates, carbonates, except for ammonium carbonate and alkali metal carbonates, are practically insoluble in water. Carbonates are important environmental bu ering agents. ey are part of the carbon dioxide/bicarbonate/carbonate bu er, which, above all, serve the world's oceans to neutralize and counterbalance acidic in uences in the environment. is bu ering system is essential in many biological processes such as maintaining a constant blood pH [8] and internal environment within homeostasis [1]: Zinc oxide ZnO at low concentration is non-toxic to human and displays good biocompatibility to human cells. ZnO showed a wide range of antibacterial activities of di erent microorganisms, including both Gram-negative and Gram-positive bacteria [9]. It was also con rmed that reinforcement of ZnO in the biocomposites introduces their antibacterial activity [10]. 130 °C, and downforce of 200 N. e coated canvas had an area weight of 220 g/m 2 and a thickness of 0.33 mm. Part of the canvas was subjected to the arti cial accelerated aging. According to standards [13,14] for accelerated aging of paper and board by dry heat treatment or moist heat treatment, part of the canvas was subjected to the so-called hot aging (at 105 °C for 144 hours), another part of the canvas was subjected to the so-called wet aging (at 80 °C and 65% of relative humidity for 144 hours). Articial aging was carried out in the WK3 climatic test room with a capacity of about 350 liters (Weiss Umwelttechnik GmbH, Reiskirchen, GE). Wet aging imitates weathering and weather in uences or storage in wet and warm environment, while hot aging imitates dry tropics or other long-term high temperature stress. It is di cult to imitate experimentally the in uence of time. e relation between accelerated and natural aging represents a serious problem. e changes monitored upon accelerated aging procedures must be correctly extrapolated to the ambient conditions. Nevertheless, in accordance with data in the literature, it is stated that three days of accelerated aging of paper and other cellulosic materials at 105 °C correspond to 25 years of natural aging [15].

Characterization of coating surfaces
e particle size used as additives and the appearance of the coated surface were scanned by optical methods. Particle size of ZnO and dolomite was measured using the particle size analyzer and laser scattering device (HORIBA LA-920, JP). e surface and cross-section of canvas were monitored using SEM scanning electron microscope VEGA TS 5130 (TESCAN, CZ) and the 3D digital multifunction microscope HIROX RH 2000 with MXB 2500REZ lens and di use adapter (HIROX, JP).
is optical method also characterized the canvas surface roughness, Rz. In accordance with standard JIS [16], Rz is the sum of the average absolute value of then height ( ve highest peaks) and the average absolute value of the depth ( ve lowest valleys from the average line of the roughness curve) on the we yarn, in microns. Ten-spot average roughness was chosen at zoom 500-x. Energy-dispersive X-ray spectroscopy (EDS), when backscattered electrons at a sampling depth of 1-2 microns characterize the chemical composition of investigated surface, is part of SEM device. EDS was chosen as a method of chemical characterization of the coating surface and percentage quanti cation of the monitored metals (Zn, Ti, Ca, Mg).

Buff ering test
25 cm 2 of each canvas including the reference sample (cotton fabric coated with only acrylic coating without additives) was immersed in 50 ml of puried deionized water for 2 days at 22 ± 2 °C to achieve an equilibrium of water with both CO 2 from the atmosphere and carbonate containing dolomite. A er 48 hours, the pH of the water in each beaker was measured. en 0.5 ml of 27 mM acetic acid was added to each beaker. All pH changes were continuously potentiometrically measured at various time intervals up to 24 hours a er application of acetic acid using glass silver chloride reference electrode.

Microbiological test
A mixture of G+ and G-bacteria containing E. coli, Staphylococcus epidermidis and Streptococcus mutans was prepared using a liquid culture medium Trypto-Soya Broth (TSB) (Biovendor, CZ) at 37 °C for 24 hours. Bacterial suspension of concentration 3 · 10 5 CFU/ml was prepared by dilution with distilled water. irty ml of this suspension was dosed into plastic tubes, samples of all four canvas were added (each with an area of 32 cm 2 ), i.e. canvas without aging, canvas a er hot aging, canvas a er wet aging, and comparative sample (canvas without addition of additives). One tube with bacterial suspension was le without the addition of canvas. All samples inserted into the bacterial suspension in tubes were shaken on a shaker at speed of 150 rpm to achieve dynamic contact of the bacterial suspension with the canvas surface. A er 4, 48 and 72 hours, 1 ml of suspension was taken from each tube, it was spotted on the surface of the agar in Petri dishes (TSA-Tryptone soya agar, Biovendor, CZ). ese samples were cultivated at 37 °C for 24 hours and then bacterial colonies were counted.

Buff ering and Antibacterial Properties of Cotton Canvas with Dolomite/ZnO-Styrene-Acrylic Complex Coating and their
Comparison with Properties after the Accelerated Aging for both dolomite and zinc oxide. While the mean of the dolomite particle size was 15.7 μm and the median of 13 μm, the powder ZnO declared by the producer as nanoparticles had a mean particle size of 25 μm (median of 24 μm) due their easy agglomeration when used in a powdered state without a dispersant. Unfortunately, the shear forces used in the standard mechanical mixing of the coating composition under laboratory conditions were not su cient to scatter ZnO agglomerates into nanoparticle sizes. As can be seen from the surface (Figure 3) and the cross-section (Figure 4) SEM images of the canvas, this particle size also re ected in a relatively high surface roughness. e Rz value was calculated as   e EDS spectra of the canvas surface were measured by SEM at the values of the middle vacuum (3-150 Pa) and was taken from two locations ( Figure 6): 1 -spot particle point (in place of surface irregularity in the coating), 2 -area (element mapping). Figure 6: Two sites of EDS analysis (spot Spectrum 1 and small area Spectrum 2) and calcium-zinc occurrence mapping on the canvas surface As shown in Figure 7 and Figure 8, for example, the zinc was detected in an amount of 31.1 wt.% (spot spectrum 1) or also 1.4 wt.% (area spectrum 2, bottom of Figure 8). e total average content of selected elements from the mapping of canvas surface is shown in Table 1.  While zinc oxide was used in an amount of 2.8 wt.% of wet coating ratio, in the dry surface layer, zinc only represents approximately 0.7 wt.%. e dolomite, which originally represented an amount of 2.1% from wet coating paste, was detected in the dry surface as calcium and magnesium in a total weight of 0.2 wt.%. Due to the weight ratio in the dolomite, where Ca and Mg represent about one third of (anhydrous) carbonate, a real weight of dolomite in the coating may be assumed to be about 0.6 wt.%.  e results of the bu ering and antimicrobial tests of the coated canvas and its two aging versions are shown by the graphs of Figure 9 and Figure 10.
e bu ering test in Figure 9 shows how the pH of the aqueous solutions with coated samples slowly returns to neutral over 15 hours. Figure 10 shows how the number of cultivated CFUs decreased. e test was run for 4 days, and bacterial suspensions with an initial concentration of 3x10 5 CFU/mL showed a di erently rapid decrease in bacterial growth. It is evident that aqueous solutions with coated canvas showed the best bu ering capacity and also the fastest decrease in bacterial growth. Simultaneously, it can be stated that even a er the simulated wet and hot aging, the canvas did not lose any of these abilities.

Discussion
e bu ering and antibacterial ability of coated canvases with both additives was best re ected in samples a er 2 days in water. A er aging that simulated about 75 years, these canvases have reduced a little all observed properties compared to the original canvas. However, the samples did not lose their antibacterial and bu ering properties. At the same time, the canvas a er hot aging (6 days at 105 °C) showed a slightly slower start, especially of antibacterial properties. is leads us to believe that even the visible and prominent particles in the coating surface are partially covered by an acrylate layer, and are essentially passivated until they are in contact with water or wet environment for a long time.
On the contrary, this may be an advantage, as particles will become most active up for longer times during long-term storage.

Conclusion
e aim of this research was to determine the buering and self-sterilizing properties of canvas with ZnO and dolomite particles in styrene/acrylic coating. ese properties were demonstrated by stress tests with samples in water. e rst was done with 27 mM acetic acid and the other using G-and G+ bacteria mixture of 3 · 10 5 CFU/mL (Escherichia coli, Staphylococcus epidemidis and Streptococcus mutans).
e canvases previously immersed for 48 hours in water bu ered the acid slowly for the next 24 hours. Also, the maximum bactericidal e ect was achieved a er 48 hours of dynamic contact of the surface of the cloths with the bacteria in the aqueous suspension. Arti cial accelerated aging slightly decreased these abilities, but canvases did not lose those properties. Hot aging, probably due to the temperature hardening of the acrylic coating, led to a slower start of the antibacterial e ect of the cloth. Despite the use of somewhat coarser particles as coating additives, we believe that the tested combination of additives is promising to produce canvas