graphite composites and its antibacterial activity at different conditions

Journal of Photochemistry and Photobiology B: Biology 151 (2015) 256–263

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ZnO/graphite composites and its antibacterial activity at different conditions Katerˇina Deˇdková a,b,⇑, Barbora Janíková c, Katerˇina Mateˇjová d, Kristina Cˇabanová a, Rostislav Vánˇa e, Aleš Kalup f, Marianna Hundáková a, Jana Kukutschová a,b a

Nanotechnology Centre, VŠB-Technical University of Ostrava, 17. listopadu 15/2172, 708 33 Ostrava-Poruba, Czech Republic Regional Materials Science and Technology Centre, VŠB-Technical University of Ostrava, 17. listopadu 15/2172, 708 33 Ostrava-Poruba, Czech Republic Department of Thermal Engineering, Faculty of Metallurgy and Materials Engineering, VŠB-Technical University of Ostrava, 17. listopadu 15/2172, 708 33 Ostrava-Poruba, Czech Republic d AGEL Laboratory, Zaluzˇanského 1192/15, 703 84 Ostrava-Vítkovice, Czech Republic e TESCAN Brno, s.r.o., Libušina trˇída 1, 623 00 Brno, Czech Republic f Department of Physical Chemistry and Theory of Technological Processes, Faculty of Metallurgy and Materials Engineering, VŠB-Technical University of Ostrava, 17. listopadu 15/2172, 708 33 Ostrava-Poruba, Czech Republic b c

a r t i c l e

i n f o

Article history: Received 29 May 2015 Received in revised form 10 August 2015 Accepted 13 August 2015 Available online 22 August 2015 Keywords: Nanostrucured composites S. aureus XRPD Raman spectroscopy Standard microdilution method

a b s t r a c t The paper reports laboratory preparation, characterization and in vitro evaluation of antibacterial activity of ZnO/graphite nanocomposites. Zinc chloride and sodium carbonate served as precursors for synthesis of zinc oxide, while micromilled and natural graphite were used as the matrix for ZnO nanoparticles anchoring. During the reaction of ZnCl2 with saturated aqueous solution of Na2CO3a new compound is created. During the calcination at the temperature of 500 °C this new precursors decomposes and ZnO nanoparticles are formed. Composites ZnO/graphite with 50 wt.% of ZnO particles were prepared. X-ray powder diffraction and Raman microspectroscopy served as phase-analytical methods. Scanning electron microscopy technique was used for morphology characterization of the prepared samples and EDS mapping for visualization of elemental distribution. A developed modification of the standard microdilution test was used for in vitro evaluation of daylight induced antibacterial activity and antibacterial activity at dark conditions. Common human pathogens served as microorganism for antibacterial assay. Antibacterial activity of ZnO/graphite composites could be based on photocatalytic reaction; however there is a role of Zn2+ ions on the resulting antibacterial activity which proved the experiments in dark condition. There is synergistic effect between Zn2+ caused and reactive oxygen species caused antibacterial activity. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Microorganisms are ubiquitous in the biosphere. Some on them do not affect humans or animals, some of them are even useful in several food productions, but there are lots of microorganisms which could have negative impact on human health. Therefore it is important to find the balance between benefits and negative effects, to have the negative effects under control. For this purpose commercially available antibiotics are being used. Nevertheless due to the huge overuse of antibiotics in the second half of twentieth century multidrug-resistant bacterial strains have originated. Researchers all over the world currently try to find new medicine to overcome these resistant strains [1]. ⇑ Corresponding author at: Nanotechnology Centre, VŠB-Technical University of Ostrava, 17. listopadu 15/2172, 708 33 Ostrava-Poruba, Czech Republic. E-mail address: [email protected] (K. Deˇdková). http://dx.doi.org/10.1016/j.jphotobiol.2015.08.017 1011-1344/Ó 2015 Elsevier B.V. All rights reserved.

Due to the unique physico-chemical properties nanomaterials are being deeply studied for their antibacterial properties in the last decade. It has been discovered that several nanomaterials exhibit strong antibacterial activity. Metal and metal oxide nanoparticles such as silver (Ag) [2], silver oxide (Ag2O), titanium dioxide (TiO2) [3], gold (Au) [4,5], silica (Si) [6] and copper oxide (CuO) [7] have been described to possess antimicrobial activity. Bare nanoparticles may pose some environmental risks due to their enhanced reactivity in comparison with bulk materials [8,9]. When nanoparticles are tightly chemically bonded to a suitable matrix (e.g. clay minerals or graphite) they still demonstrate unique e.g. antibacterial properties but their environmental risks are decreased due to the limited mobility in the environmental media. Graphite is layered structured allotrope carbon made from stacked graphene sheets. It exhibits anisotropy in electrical and mechanical properties. It is a common and widely used material

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with relatively low damaging effects for human health or the environment. In this work graphite was chosen as a matrix for the nanostructured composite material due to its wide use and environmental safety. The aim of the study was to prepare, characterize ZnO/graphite nanostructured composite material and explore its antibacterial activity against selected human pathogens in relation to the exposure to daylight irradiation in comparison with dark conditions. 2. Materials and methods 2.1. Studied nanocomposites ZnCl2 p.a. and Na2CO3 (Lachema) were used for the synthesis of ZnO precursor. Two graphite substrates, micromilled – further assigned as Gra(1) and high purity natural graphite – further assigned as Gra(2) (Graphite Ty´n, spol. s.r.o., Czech Republic), were used as matrices for ZnO precursor nanoparticles. The synthesis includes two main parts. In the first step, the ZnO precursors, Na2Zn3(CO3)4
(H2O)3 and Zn5(OH)6(CO3)2, was prepared by the reaction of ZnCl2 with Na2CO3 saturated solution stirring at room temperature in the presence of graphite substrate. During the second step, the ZnO precursor was dried at 100 °C and thermally decomposed at 500 °C to form ZnO nanoparticles. Composites with 50 wt.% of ZnO were prepared and designated as ZinGra(1)5X and ZinGra(2)5X, where X corresponds to thermal treatment temperature (1 – drying at 100 °C and 5 – calcination at 500 °C) [10]. 2.2. Microscopic and phase analysis The prepared samples were sintered in an electrical laboratory furnace LH15/13 (LAC, s.r.o.) with the heating rate of 5 °C min1 up to the final sintering temperature of 1000 °C and keeping that temperature for 1 h. The weight after burning (i.e. the weight of pure ZnO) divided to the weight of the sample before burning gives the weight percents of ZnO determined. The TG/DTA analysis was performed on Setaram SETSYS 18TM device. Both samples had approximately 22 mg and they were measured in Al2O3 crucibles under air atmosphere with heat rate 5 C min1. The diffractometer Bruker D8 Advance (Bruker AXS, Germany) equipped with detector VÅNTEC 1 was used to record the XRPD patterns under Co Ka irradiation (k = 1.789 nm). During the measurement the reflection mode was used and powder samples were pressed in a rotational holder. The database PDF 2 Release 2004 (International Centre for Diffraction Data) was used to evaluate the phase composition. Smart Raman Microscopy System XploRATM (HORIBA Jobin Yvon, France) which allows point analysis was used for the phase characterization of the prepared nanocomposites. Raman spectra were acquired with 532 nm excitation laser source, with 50 objective and using 1200 gr./mm grating. Scanning electron microscope MAIA3 GMU (TESCAN) was used – ultra-high resolution SEM with Schottky field emission cathode. Images were taken by using a combination of InBeam SE + LowEnergy BSE detector at 2.5 kV. EDS analysis was performed with X-MaxN 150 (Oxford instruments) and the EDS data were processed in AZtec software. 2.3. Antibacterial assessment Four different human pathogenic bacterial strains were used for the in vitro determination of antibacterial activity of the prepared samples. Glucose broth (HiMedia) was used as a growth media.

257

Turbidity of the inoculums was measured using DEN-1 McFarland Densitometer (BioSan). Incubation of bacteria was conducted in Biological thermostat BT 120 M at 37 °C. Standard microdilution method enabling determination of the minimum inhibitory concentration (MIC) of tested substances served as the method for evaluation of the antibacterial activity of the ZinGra composites. Disposable microtitration plates were used for the testing. Commercial solid blood agar plates for the cultivation of bacteria without any additional modifications were used. Liquid growth media were prepared according to producer’s instructions and sterilized in an autoclave. Suspension of the ZinGra samples in the growth media was diluted to achieve the following concentrations of 100, 33.3, 11, 3.7, 1.2, 0.41, 0.014 mg/ml of ZinGra in the media. Staphylococcus aureus 3953, Enterococcus faecalis 4224 and Pseudomonas aeruginosa 1960 were acquired from the Czech Collection of Microorganisms (Czech Republic). The used bacterial inoculums had the following cell concentration of 1.1  109 (S. aureus), 1.3  109 (E. faecalis) and 1.1  109 (P. aeruginosa) CFU/ml (colony-forming units per milliliter). Each compartment of the microtitration plates was inoculated. This plate is called the reaction plate. The lamp with wide spectrum bulb with intensity of 2.4 mW/cm2, which was already used in our previous experiments [11] was placed 10 cm above the reaction plate to induce photo activation of the ZinGra samples, and 8 h of irradiation of the plate was applied on the first day of the experiment. Parallel reaction plate with the same composites at the same concentrations was performed at dark conditions without any irradiation. After the defined time period present living bacterial cells were transferred from the reaction plates to the pure growth media using an inoculation hedgehog. These re-inoculated plates were incubated at 37 °C for 24 h and then the MIC values were determined according to visible growth inhibition. On the second day living bacterial cells from the reaction plate were transferred to a plate with pure media, 8 h of irradiation the same approach was applied again. After the irradiation the cells were transferred from the reaction plate to the plates with pure media. The non-irradiated reaction plate was performed at the dark conditions however the same defined time period as in the case of irradiated one was followed. On the third day the experiment continued in the same manner. Before and after the irradiation living cells were transferred from the reaction plate to a plate with pure media and 8 h of irradiation was applied again. This modification of the standard microdilution method eliminated issues with the determination of MIC caused by turbidity of dead cells or caused by the presence of the ZinGra sample particles in microtitration plate, because only living bacterial cells can be captured by needles of the inoculation hedgehog.

3. Results and discussion Graphite is not stable at oxidative atmosphere at 1000 °C, it burns provably. Therefore this fact was used for determination of the content of ZnO in the composites. The weight after burning (the weight of pure ZnO) divided to the weight of the sample before burning gives the weight percent of ZnO determined (Table 1). Pure Zn5(OH)6(CO3)2 has maximal mass loss at 247 °C [12], ZinGra samples have maximal mass loss at 239 °C (Fig. 1) and 244 °C Table 1 Determined content of ZnO in the ZinGra composites. Sample

Content of ZnO [wt.%]

ZinGra(1)55 ZinGra(2)55

42.43 45.44

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258

TG/mg

HeatFlow/µV Exo

40

-2,29 %

35

2 -11,43 %

30 25

0

20 -2

15 10

-4

5

-34,25 %

0 -6

-5 121 °C

-10 240 °C

100

200

300

400

500

600

700

800

Sample temperature/°C

Fig. 1. The TG (green) and DTA (blue) curves of ZinGra(1)55. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

HeatFlow/µV

TG/mg

Exo 4

-2,27 %

35 30 25

-11,53 %

2

20 15

0

10 -2

5 -36,56 %

0 -4 -5

-6

-10

118 °C 245 °C

100

200

300

-15 400

500

600

700

800

Sample temperature/°C

Fig. 2. The TG (green) and DTA (blue) curves of ZinGra(2)55. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(Fig. 2), respectively. This small shift to lower temperatures is caused due to the presence of ‘‘impurities” (graphite, Na+ compound . . .). Enormous weight loss and big exothermic peak above 500 °C is recorded due to the burning of graphite. This process is slow and gradual, where weight loosing occurs. Deformation of this big peak is due to small endothermic peak. This endothermic peak is probably present due to the decomposition of traces of NaOH and/or Na2CO3. The XRPD patterns of the ZinGra(1) and ZinGra(2) composites are shown in Figs. 3 and 4. Diffraction patterns of the dried composites ZinGra(1)51 and ZinGra(2)51 confirmed the origination of Na2Zn3(CO3)4
(H2O)3 (PDF card no. 01-0457) as a result of ZnCl2 and Na2CO3 reaction. Moreover, presence of Zn5(OH)6(CO3)2 (PDF card no. 54-0047) was confirmed in the XRPD patterns of these dried samples. The carbon matrices for anchoring of ZnO contain elemental carbon (PDF card no. 74-2328) and graphite (PDF card no. 56-0159).

After the calcination at the temperature of 500 °C, the ZnO formation (PDF card no. 03-0888) from precursors is confirmed in samples ZinGra1(55) and ZinGra2(55). The reflections of graphite are still present in the XRPD patterns of these calcined samples. Probably, not all precursors were decomposed to the ZnO particles. XRPD patterns of calcined samples shows some reflections which may be ascribed to some residual of precursors. The ZnO crystallite sizes were calculated according the Sherrer formula [14] from the (1 0 1) ZnO reflection (2h = 42.4°) as 30 nm for ZinGra(1)55 and 27 nm for ZinGra(2)55. Recorded Raman spectra can be seen at Fig. 5. The presence of graphite is visible due to the bands around 1575 cm1 (G-band) and 2700 cm1 (historically designated as the G0 -band) [13,14] in all ZinGra samples. Main carbonate band around 1076 cm1 is present in all ZinGra samples, whereas in the sample ZinGra(2)51 it has the highest intensity. Thus, the other carbonate bands are clearly visible at 114, 145, 249, 390 and 777 cm1. Bands around

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Fig. 3. XRPD patterns of the ZinGra(1) composites: 1 – graphite, 2 – Na2Zn3(CO3)4
(H2O)3 precursor, 3 – Zn5(OH)6(CO3)2, 4 – carbon, 5 – ZnO.

Fig. 4. XRPD patterns of the ZinGra(2) composites: 1 – graphite, 2 – Na2Zn3(CO3)4
3(H2O) precursor, 3 – Zn5(OH)6(CO3)2, 4 – carbon, 5 – ZnO.

100, 325 and 430 cm1 are present in the samples ZinGra(1)51, ZinGra(1)55 and ZinGra(2)55 and may be attributed to the presence of ZnO in the composites. The absence of these bands in the ZinGra(2)51 could be based on the absence of ZnO particles in the analyzed spot caused by the non-homogeneous distribution of ZnO in the composites. XRD did not prove ZnO in ZinGra(1)51 however Raman microspectroscopy is point analysis and it might be possible that in analyzed spots of ZinGra(1)51 a small amount of ZnO occurred and it is concurrently under the detection limit of XRD. The SEM images of the studied samples (Figs. 6 and 7) revealed the matrix consisting of micro-sized graphite particles having a layered structure with sub-micron clusters of ZnO precursors (Figs. 6A and 7A) or ZnO particles (Figs. 6B and 7B) attached onto the surface of the graphite matrix. Distribution of ZnO particles on the surface of the graphite matrix is not homogenous. Nevertheless, there is a difference in homogeneity among the composites with micromilled graphite (Gra(1)) and pure natural graphite (Gra(2)), where the distribution of ZnO particles is more homogenous in the case of Gra(1).

Figs. 8 and 9 show EDS maps of elemental composition of selected areas of the ZinGra samples. The presence of carbon, oxygen and zinc was confirmed. It is evident, that the clusters of ZnO are not homogenously dispersed on the graphite sheets. There is also a difference between the samples dried at 100 °C (Figs. 8A and 9A) where bigger clusters of ZnO precursors are observable. On the contrary, in the case of the samples calcined at 500 °C (Figs. 8B and 9B) clusters of ZnO are not that big and are more homogenously dispersed on the graphite sheets. Antibacterial activity, expressed as the MIC values (the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation) of the ZinGra samples was evaluated using three bacterial strains. Values of MIC for the ZinGra samples against all bacterial strains are summarized in (Table 2). Pure graphite did not exhibit antibacterial activity; therefore MIC could not be determined and are not included in the tables. When the MIC values could not be determined for the ZinGra composites, it could be caused by the MIC value being higher than the concentration range used. This maximal concentration of the applied materials was selected to be 100 mg/ml due to

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Fig. 5. Raman spectra of all ZinGra composites.

the fact that the tested materials are not pure nanoparticles but composites where the amount of effective component (ZnO) is around 50 wt.%. There is a visible difference between the onsets of the daylight induced antibacterial activity of all samples and the antibacterial activity under dark conditions against S. aureus; where the irradiated composites exhibited faster onset of the antibacterial activity than the non-irradiated ones. The same pattern is visible in the case of ZinGra(1)55 and ZinGra(2)55 against E. coli and P. aeruginosa. Significant difference between irradiated and non-irradiated antibacterial activity of the composites ZinGra(1)51 and ZinGra (2)55 against E. coli and P. aeruginosa was not observed. Generally lower values of MIC achieved irradiated composites in comparison with non-irradiated. The extension of reaction time causes decrease of MIC values. The lowest MIC values were achieved for irradiated ZinGra(2)55 against S. aureus (0.41 mg/ml). There are several proposed mechanisms of the antibacterial activity of ZnO nanoparticles. One of them is based on the release of Zn2+ ions and consequent diffusion of these ions into the cytoplasm [15]. Other is based on the reaction of ROS with bacterial cells due to the fact that ZnO belongs to the group of semiconductor materials with wide band gap. The study of the antibacterial activity of ZnO of Zhang [16] proposed mechanism of antibacterial activity based on the direct contact of nanoparticles with the cell wall, their

Fig. 6. Examples of SEM images of ZinGra(1)51(A) and ZinGra(1)55(B) particles.

Fig. 7. Examples of SEM images of ZinGra(2)51(A) and ZinGra(2)55(B) particles.

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Fig. 8. Selected areas of the ZinGra(1)51(A) and ZinGra(1)55(B) sample and its corresponding EDS maps of elemental composition.

Fig. 9. Selected areas of the ZinGra(2)51 and ZinGra(2)55 samples and its corresponding EDS maps of elemental composition.

deposition on the cell wall which leads to changes in the permeability of cell wall. The results obtained in this study indicates the synergic effect of the daylight-induced production of ROS and antibacterial activity under dark conditions probably caused by release of Zn2+ ions into growth media. Explanation of the difference between onset of dried and calcined samples can be also explained due to the presence (calcined samples) or absence (dried samples) of ZnO. In the case of calcined samples, the photocatalytic reaction is proved and role of Zn2+ ions is secondary. In the case of dried sample, only precursor of ZnO is present and therefore the mechanism is based only on the Zn2+ mechanism. Our previous study described the daylight-induced antibacterial activity of composite ZnO/kaoline (ZinKa) [17]. The obtained MIC values are nearly similar to the ZinGra composites; however the antibacterial

activity of the ZinGra composites to P. aeruginosa is slightly higher than of ZinKa. It should be mentioned that the matrix in a nanostructured composite material could play a role in resulting biological activity. Our studies proved that kaoline or graphite can be used for anchoring of ZnO nanoparticles to decrease their possible mobility in the environment. From this perspective it has to be concluded that the nanocomposites including ZnO do not cause inhibition of bacterial growth in the wide range as several antibiotics and there are several factors affecting the resulting biological properties. The outcome of the day light-induced antibacterial activity of the composites ZinGra is relevant in terms of potential applications of these nanocomposites for antibacterial modification of various surfaces. These nanocomposites would be used in future for surface treatment for reduction of potential infection.

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262 Table 2 Experimental MIC values (mg/ml). Reaction time

ZinGra(1)51 S. aureus

ZinGra(1)55 P. aeruginosa

S. aureus

Dark

Light

Dark

E. coli Light

Dark

Light

Dark

Light

E. coli Dark

Light

Dark

P. aeruginosa Light

30 min 60 min 90 min 120 min 180 min 240 min 300 min 1 day before irradiation 1 day after irradiation 2 day before irradiation 2 day after irradiation 3 day before irradiation 3 day after irradiation

>100 >100 >100 >100 >100 >100 >100 33.3 33.3 33.3 33.3 33.3 33.3

>100 >100 >100 >100 100 100 100 33.3 11.1 11.1 11.1 11.1 11.1

>100 >100 >100 >100 >100 >100 >100 100 100 100 100 100 100

>100 >100 >100 >100 >100 >100 >100 100 100 100 33.3 33.3 33.3

>100 >100 >100 >100 >100 >100 >100 100 100 100 100 100 100

>100 >100 >100 >100 >100 >100 100 100 100 100 100 100 100

100 100 100 100 100 33.3 33.3 11.1 11.1 11.1 11.1 11.1 11.1

>100 100 100 33.3 33.3 33.3 33.3 11.1 3.7 3.7 3.7 3.7 3.7

>100 >100 >100 >100 >100 >100 >100 33.3 33.3 33.3 33.3 33.3 33.3

>100 >100 >100 100 100 33.3 33.3 33.3 33.3 11.1 11.1 11.1 11.1

>100 >100 >100 >100 >100 100 100 100 100 100 100 100 100

>100 100 100 100 100 100 33.3 33.3 33.3 33.3 33.3 33.3 33.3

30 min 60 min 90 min 120 min 180 min 240 min 300 min 1 day before irradiation 1 day after irradiation 2 day before irradiation 2 day after irradiation 3 day before irradiation 3 day after irradiation

ZinGra(2)51 >100 >100 >100 >100 >100 >100 >100 >100 >100 33.3 >100 33.3 >100 33.3 11.1 11.1 11.1 11.1 11.1 11.1 11.1 11.1 11.1 11.1 11.1 11.1

>100 >100 >100 >100 >100 >100 >100 100 100 33.3 33.3 33.3 33.3

>100 >100 >100 >100 >100 >100 100 100 100 33.3 33.3 33.3 33.3

>100 >100 >100 >100 >100 >100 >100 100 100 100 100 100 100

>100 >100 >100 >100 >100 >100 >100 100 100 100 100 100 100

ZinGra(2)55 >100 >100 >100 >100 >100 >100 >100 >100 >100 11.1 100 11.1 100 11.1 11.1 11.1 11.1 0.41 11.1 0.41 11.1 0.41 11.1 0.41 11.1 0.41

>100 >100 >100 >100 >100 >100 >100 33.3 33.3 33.3 33.3 33.3 33.3

>100 >100 >100 100 100 100 33.3 33.3 33.3 11.1 11.1 11.1 11.1

>100 >100 >100 >100 >100 100 100 100 100 100 100 100 100

>100 >100 100 100 100 100 100 11.1 11.1 11.1 11.1 11.1 11.1

4. Conclusions

References

The ZnO/graphite composites were laboratory prepared by the thermal decomposition of Na2Zn3(CO3)4
(H2O)3 and Zn5(OH)6 (CO3)2, which are products of the reaction of zinc chloride and Na2CO3 in aqueous suspension with graphite, while natural and micro-milled graphite were used as a matrix. XRPD proved formation of zincite during the thermal decomposition of Na2Zn3(CO3)4
(H2O)3 and Zn5(OH)6(CO3)2 at 500 °C. Scanning electron microscopy confirmed the presence of ZnO particles and proved that ZnO particles are anchored onto the surface of graphite. EDS mapping in connection with SEM revealed that clusters of ZnO are not homogenously distributed on the graphite matrix. Antibacterial assays using common human pathogen bacterial strains showed that the ZinGra samples have antibacterial potency under dark conditions and under the daylight irradiation where the onset of the antibacterial activity is faster under the irradiation. The lowest MIC was achieved against S. aureus for ZinGra(2)55 after the daylight irradiation. Composites ZinGra could find potential applications e.g. in the field of antibacterial modification of surfaces of various materials, when its activity was observed under daylight irradiation hence UV light is not necessary to induce the antibacterial activity of the ZinGra composites. Moreover, these composites may pose lower environmental risks due to the bonding of ZnO nanoparticles to the graphite matrix.

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Acknowledgement This paper was created on the Faculty of Metallurgy and Materials Engineering in the Project No. LO1203 ‘‘Regional Materials Science and Technology Centre – Feasibility Program” funded by Ministry of Education, Youth and Sports of the Czech Republic and supported by project reg. no. SP2015/54 (Ministry of Education, Youth and Sports of the Czech Republic).

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