Hybrid Ag0/Ag2CO3-eggshell-plant nanocomposites for antimicrobial action prepared by bio-mechanochemical synthesis

Vol.:(0123456789) 1 3 Applied Nanoscience (2022) 12:1899–1916 https://doi.org/10.1007/s13204-022-02417-6 ORIGINAL ARTICLE Hybrid Ag0/Ag2CO3–eggshell–plant nanocomposites forantimicrobial action prepared bybio‑mechanochemical synthesis MatejBaláž1 · MarianoCasas‑Luna2· AdrianAugustinyak3,4· ĽudmilaTkáčiková5· KamilSzmuc6· MáriaKováčová1· LadislavČelko2· YaroslavShpotyuk7,8 Received: 15 December 2021 / Accepted: 6 February 2022 / Published online: 28 February 2022 © King Abdulaziz City for Science and Technology 2022 Abstract Silver carbonate and elemental silver nanoparticles embedded in the matrix of biological materials were successfully prepared via a bio-mechanochemical synthesis, i.e., one-step solvent-free approach utilizing high-energy ball milling. Namely, the powders of silver nitrate as a precursor of silver and a mixture of natural materials (eggshell wasteand Thymus serpyllum L. plant) powders as reducing and stabilizing agents, were milled together. The optimization of experimental conditions was performed using a Taguchi design. The presence of nanoparticles was confirmed by a bunch of experimental techniques including XRD and TEM, their size being up to 15nm. By means of AAS, the level of Ag stabilization was determined, being better when eggshell content was higher. The products alsoexhibited the antibacterial activity, and although the agar well diffusion method did show better results for the nanoparticles prepared usingthe eggshell or the plant alone, the respiratory tests have shown a good response also for the product prepared using a combination of the two. The strongest respiratory inhibition against Escherichia coli was observed for the nanoparticles prepared using only eggshell, but the ones synthesized using both reducing agents caused a prolonged inhibition against Staphylococcus aureus. The proposed synthetic approach offers the use of environmentally harmless technique and natural materials to yield antibacterially active nanoparticles. Keywords Mechanochemistry· High-energy ball milling· Silver· Silver carbonate· Nanoparticles· Antibacterial activity· Eggshell· Plant Introduction Silver nanoparticles are still a hot topic because of their multidisciplinary application (Verma and Maheshwari 2019; Beyene etal. 2017). Mainly their antibacterial action is of particular interest (Marambio-Jones and Hoek 2010; Ahmed etal. 2016). Many methods have been used for their preparation (Iravani etal. 2014), among which the biological synthesis became the mostly used one in recent years due to its environmental friendliness and low cost (Sharma etal. 2009; Some etal. 2019). Nevertheless, also this method has its drawbacks, mainly being a multistage process (the preparation of extract and a separate synthesis step is necessary) and usually the reaction runs at elevated temperatures. Moreover, the stability of the prepared nanosuspension is often questionable (Srikar etal. 2016; Baláž etal. 2017a). To tackle this, our research group proposed a one-step solid-state alternative entitled bio-mechanochemical synthesis (Baláž etal. 2017b). So far, we have shown that it can be used for various plants (Baláž etal. * Matej Baláž [email protected] 1 Institute ofGeotechnics, Slovak Academy ofSciences, Watsonova 45, 04001Kosice, Slovakia 2 Central European Institute ofTechnology, Brno University ofTechnology, Purkynova 123, 61200Brno, CzechRepublic 3 Chair ofBuilding Materials andConstruction Chemistry, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355Berlin, Germany 4 Faculty ofChemical Technology andEngineering, The West Pomeranian University ofTechnology inSzczecin, Piastów Avenue 42, 71-065Szczecin, Poland 5 Department ofMicrobiology andImmunology, University ofVeterinary Medicine andPharmacy, Kosice, Slovakia 6 Institute ofMaterials Engineering, University ofRzeszow, 1, Pigonia Str., 35-959Rzeszow, Poland 7 Institute ofPhysics, University ofRzeszow, 1, Pigonia Str., 35-959Rzeszow, Poland 8 Ivan Franko National University ofLviv, 1, Universytetska Str., Lviv79000, Ukraine 1900 Applied Nanoscience (2022) 12:1899–1916 1 3 2017b, 2021a; Kováčová etal. 2020) and lichens (Baláž etal. 2020; Goga etal. 2021). In general, mechanochemistry utilizing tools of high-energy ball milling is suitable, among others, also for the production of inorganic nanoparticles (Baláž etal. 2013) and for the treatment of natural materials, including waste (Baláž 2021). The scalability of the mechanochemical processing is a hot topic today, and methods like wet stirred media milling (Mucsi 2019), twin screw extrusion (Crawford etal. 2017) and eccentric vibratory milling (Baláž etal. 2021b) are great candidates for a large-scale implementation. Thymus serpyllum L. plant, belongs among the most common plants in the world and has been efficiently used for the synthesis of Ag NPs in the past (Erci and Torlak 2019). Moreover, also the bio-mechanochemical synthesis using this plant has been successfully performed in Kováčová etal. (2020), showing it to be the most suitable candidate out of the three plants investigated in the mentioned study. During the green synthesis, very often only a plant extract is used to serve the roles of both reducing and stabilizing agents. However, there are couple of studies, where another natural and cheap material (e.g., eggshell waste) can be used to improve the result. Specifically, eggshell has been used in combination with the plant Centella asiatica L. in Pham etal. (2020) or Cacumen platycladi in Huang etal. (2020). In the latter case, the eggshellderived hydroxyapatite–Ag nanoparticles nanocomposite was prepared. The authors proposed that eggshell serves as a stabilizer of small metallic nanoparticles that reduces their aggregation. Eggshell waste is one of the most abundant wastes coming from food industry and has been used in the past in the process of Ag NPs production, namely as a support for bio-inspired synthesis (Liang etal. 2014; Liu etal. 2016; Cai etal. 2019; Huang etal. 2021; Raz etal. 2019), or directly as a reducing agent (Tiimob etal. 2017; Apalangya etal. 2014; Huang etal. 2016; Du etal. 2017). Sometimes, also elevated temperatures were used to yield the nanocomposite with interesting properties (Alsohaimi etal. 2020; Guo etal. 2019). High-energy ball milling has also been applied to obtain Ag NPs using eggshell in the past (Baláž etal. 2017b; Tiimob etal. 2017; Apalangya etal. 2014). The aim of this paper was to investigate a possible synergistic effect between the plant and the eggshell in the synthesis/stabilization of Ag nanoparticles. This is the first report on a mixture of two reducing/stabilizing agents for the production of Ag NPs using mechanochemistry. Moreover, the metathesis reaction between Ag from AgNO3 and Ca from CaCO3 present in the eggshell was discovered and the antibacterial activity of Ag/Ag2CO3 composite is reported herein, which is also a novel viewpoint. Materials andmethods Materials AgNO3 (99.9% purity from Centralchem, Slovakia) was used without any further purification. Thymus serpyllum L. plant (SER) was purchased from a local company (Agrokarpaty s.r.o., Slovakia) concerned with a cultivation of medicinal plants. Prior to high-energy milling experiments, the whole plant was milled in a kitchen mixer (type HR2061, Philips, Netherlands) and sieved to reach the particle size ≤ 1mm. Eggshell was provided by local canteen in Košice. It was washed with distilled water to remove residual albumen, dried and then micronized using kitchen mixer (type HR2061, Philips, Netherlands) and sieved to reach particle size smaller than 1mm. The eggshell membrane was not separated from eggshell in this case. Mechanochemical synthesis andwashing During the mechanochemical synthesis, a total solid mass of 3.0g was milled in a Pulverisette 7 Premium line planetary ball mill (Fritsch, Germany) under the following conditions: air atmosphere, 15 tungsten carbide (WC) milling balls (10mm diameter), ball-to-powder ratio 37, milling speed 500rpm and milling time ranging from 15 to 120min. After each 30min of milling, 15-min break was done to avoid overheating. After milling, 1.0g sample of the as-obtained powder was washed Table 1 Experimental plan based on Taguchi design for the optimization of experimental conditions using ES/SER mixture as reducing agent Sample AgNO3 mass (g) Eggshell (ES)/Thymus serpyllum, L. (SER) ratio Milling time (min) Ag-ES-SER T1 0.1667 0.333 15 Ag-ES-SER T2 0.1667 1.000 45 Ag-ES-SER T3 0.1667 3.000 120 Ag-ES-SER T4 0.5000 0.333 45 Ag-ES-SER T5 0.5000 1.000 120 Ag-ES-SER T6 0.5000 3.000 15 Ag-ES-SER T7 1.5000 0.333 120 Ag-ES-SER T8 1.5000 1.000 15 Ag-ES-SER T9 1.5000 3.000 45 1901Applied Nanoscience (2022) 12:1899–1916 1 3 with 50mL distilled water, stirred for 30min and subsequently filtered to remove non-reacted AgNO3. Experimental setup To optimize the process, three factors were investigated, namely AgNO3 amount, eggshell/T. serpyllum L. (ES/ SER) ratio and milling time (Table1). The experiments were designed using aMINITAB14 software (Minitab, Ltd., United Kingdom). The levels of factors dealing with the milling process were selected to be in the range which is usually applied for mechanochemical experiments. The overall mass of the reaction mixture was always3.0g, and the amount of introduced AgNO3 was 0.1667g (6 wt%), 0.5g (17 wt%), or 1.5g (50 wt%). The rest of weight was represented by the ES/SER mixture (the exact masses of the eggshell and T. serpyllum L. plant are provided in TableS1 in the Supplementary file). The weight balance can be also presented in the form of ES/SER-to-AgNO3 ratio and the values are 16.99, 5.00 and 1.00 for the reaction mixtures containing 0.1667, 0.5 and 1.5g AgNO3, respectively. Characterization methods The phase composition of all Ag-SER-EST samples was analyzed by X-ray diffraction (XRD, Rigaku SmartLab 3kW). The diffractograms were acquired in a Bragg–Brentano geometry between 2θ angles of 15°–70° with a scan speed of 4° per minute. The X-ray source was generated using a CuKα (λ = 0.15418nm) radiation at an operating voltage and current of 30kV and 40mA, respectively. The phase identification was performed using the Inorganic Crystal Structure Database (ICSD) and the quantitative analysis was done by Rietveld refinement using the HighScore Plus 3.0 software (Panalytical B.V., The Netherlands). Fitting of the diffractograms was performed using a fifth-grade polynomial background and a pseudo-Voight peak profile. All fitting parameters were varied until the lowest possible residual of least-squares value (Rp) a goodness of fit (GOF) were reached. Crystallite sizes were calculated by Scherrer equation from the main diffraction peaks of each phase present in the diffraction patterns. The XRD patterns of the SER-, and eggshell-free analogues were measured using a D8 Advance X-ray diffractometer (Bruker, Germany) in the Bragg–Brentano geometry, working with a CuKα (λ = 0.15418nm) radiation and a scintillation detector. The operating voltage and current were 40kV and 40mA, respectively. All samples were scanned from 15° to 70° with steps 0.05° and 15-s counting time. The concentration of silver and calcium in solid samples was analyzed using an atomic absorption spectrometer SPECTRAA L40/FS (Varian, Australia). The microstructure and morphology of obtained samples were studied using the transmission electron microscopy (S/ TEM) FEI Tecnai Osiris equipped with field emission gun (FEG), operating at accelerating voltage 200kV. Samples were examined in classical transmission (Bright Field); high resolution (HRTEM) and selected area diffraction (SAED) modes. A chemical composition mapping was performed by a super energy-dispersive X-ray spectrometer (EDS) coupled with TEM. Antibacterial activity Agar well diffusion The antibacterial properties of the samples were evaluated by the agar well diffusion method by a slight modification of the process reported in Rojas etal. (2006). The tested bacteria (Escherichia coli CCM 3988) were obtained from the Czech collection of microorganisms (CCM). The procedure was as follows: Escherichia coli were kept frozen at −20 °C in LB medium (Sigma-Aldrich, Saint-Louis, MO, USA) with 20% v/v glycerol. Before the experiments, bacteria were revived in LB medium and incubated at 37 °C with agitation (160rpm) for 20h and used as the source of inoculum for each experiment. The inoculum from this overnight culture was prepared by adjusting the density of culture to equal that of the 0.5 McFarland standard (1–2 × 108CFU/mL) by adding a sterile saline solution. These bacterial suspension was diluted in a ratio 1:300 in aliquid plate count agar (HIMEDIA, Mumbai, India) resulting in the final concentration of bacteria of approximately 5 × 105CFU/mL, and 20mL of this inoculated agar was poured into aPetri dish (90mm diameter). Once the agar was solidified, 5 mm diameter wells were punched in the agar and filled with 50 µL of samples prepared in the form of suspensions (20mg of the samples were dispersed in 1mL of distilled water). Gentamicin sulfate (Biosera, Nuaille, France) with a concentration of 50μg/mL or 30μg/mL was used as a positive control. The plates were incubated for 20h at 37°C. Afterwards, the plates were photographed and the inhibition zones were measured by the ImageJ software. The values used for the calculation are mean values calculated from 3 replicate tests. 1902 Applied Nanoscience (2022) 12:1899–1916 1 3 The antibacterial activity was calculated by applying the formula reported in Rojas etal. (2006): where RIZD is the relative inhibition zone diameter (%) and IZD is the inhibition zone diameter (mm). As a negative control, the inhibition zones of distilled water equal to 0 were taken. The inhibition zone diameter (IZD) was obtained by measuring the diameter of transparent zone and subtracting the size of the wells (5mm). Optical density ofcultures andrespiration studies Two bacterial models(Escherichia coli ATCC ® 25922™ and Staphylococcus aureus ATCC ® 33591™)were used to test the influence of the nanoparticles. The microorganisms were kept frozen at −20°C in trypticasein soy broth (TSB, Biomaxima, Lublin, Poland) medium with 20% v/v glycerol. Bacteria were revived on trypticase soy agar (TSA, Biomaxima, Lublin, Poland) medium and incubated at 37°C prior to use in the experiments. Three final products were used in this part of the experiments. Suspensions in deionized water were sterilized at 100°C for 15min, cooled down to ambient temperature, and sonicated in a water bath sonicator (35kHz) for 30min to improve dispersion. The sample concentrations tested in the analyses were 25µg/mL 50µg/mL, and 100µg/mL. For 24-h toxicity test, bacteria were inoculated 1:500 from overnight culture (14–16h old) to a fresh TSB medium and incubated at 37°C and agitated (150rpm). The optical density (λ = 600nm) was recorded at time 0 and 24h on BioTek Synergy H1 (Winooski, VT, USA). Afterward, 10% v/v of resazurin (1mg/mL, Merck, Darmstadt, Germany) was added to the cultures, the samples were incubated at ambient temperature for five minutes, and fluorescence was recorded (λex = 520nm, λem = 590nm). The cultures for respiration monitoring during the logarithmic growth phase were prepared in the same manner as for the 24-h toxicity test. After 2-h incubation 10% of resazurin was added. The fluorescence (λex = 520nm, λem = 590nm) was measured for 4h in 10-min intervals. All the samples in the 24-h assay were prepared in 24 repetitions. The ones included in the respiration monitoring assay were prepared in eight repetitions. The differences between samples were analyzed with one-way ANOVA performed in Origin 2021 software (OriginLab Corp., Northampton, MA, USA). Comparisons were done by Turkey’s post hoc test, where the results with p < 0.05 were accepted as significantly different. %RIZD = [(IZD sample−IZD negative control) ∕IZD gentamicin]×100, Results anddiscussion X‑ray diffraction ofas‑received powders As a first measure of the synthesis success, the XRD patterns of the as-received powders were measured (Fig.1). Basically, the prepared samples can be divided into two groups. For the samples T1–T6, the XRD patterns are dominated by the peaks of calcite (as a result of the presence of eggshell which contains around 94% CaCO3) and also diffractions corresponding to elemental silver can be found in different amounts. The largest amount of silver seems to bepresent in the sample T5, followed by T3. In these two cases, the milling time was 120min, so the longer milling seems to favor the formation of elemental silver. This is in correlation with the results obtained for bio-mechanochemical synthesis using lavender (Baláž etal. 2021a). The XRD patterns of the second group of samples T7–T9 are dominated by the peaks corresponding to non-reacted AgNO3 and CaCO3, however, the traces of elemental Ag can also be noticed. For the sample T7 (milled for 120min), the amount of Ag0 is, again, substantial. The presence of non-reacted AgNO3 in T7–T9 samples is due to the fact that it represented 50% of the starting reaction mixture and therefore, the introduced amount of reducing agents was not satisfactory to reduce all of it. In general, both reducing agents are capable of reducing Ag to its elemental form. For SER plant, two hours were Fig. 1 XRD patterns of the as-received Ag-ES-SER T1–T9. At the bottom, the positions and intensities of identified crystallographic phases according to ICSD database are provided for comparison: calcite 166,364; silver 180,878; silver carbonate 93,988; silver nitrate 201,605 1903Applied Nanoscience (2022) 12:1899–1916 1 3 satisfactory for all AgNO3 diffractions to disappear from XRD pattern under identical conditions in Kováčová etal. (2020). With regards to eggshell, the eggshell membrane (ESM) is well-capable of reducing AgNO3 to elemental Ag in 30min as shown in Baláž etal. (2020). We are of the opinion that eggshell alone does not have the ability to reduce it, but if the ESM is not separated from it, the reduction can be observed as in Tiimob etal. (2017), where the authors purchased the eggshell from a commercial company and it is possible that it was not purified from ESM. In that case, milling time of minimum 5h was reported. The real separation of ESM from ES was also not done in Apalangya etal. (2014), where similarly to our case, the reduction was observed. The present results show that the realization of the studied reactions in labscale planetary ball mills is possible, and similarly to other systems (Baláž etal. 2019a, b, 2018; Achimovičová etal. 2019), might be suitable for a scale-up implementation, e.g., by eccentric vibratory milling. The mechanism of Ag NPs formation using biological materials as both reducing and stabilizing agents is based on the reduction of Ag(I) ion into elemental silver Ag(0) (Akintelu etal. 2020). Most often, the main role in the reduction process is ascribed to hydroxyl (–OH) groups, which are present in various biomolecules like amino acids, enzymes, flavonoids, etc. (Akintelu etal. 2020; Das and Brar 2013). In the present case, there are two reducing agents—T. serpyllum L. plant and eggshell. In the case of the plant, an example of the hydroxyl group-containing compound might be quercetin. With regards to eggshell, its proteinaceous component—eggshell membrane (ESM) should be responsible for the reduction. Recent works have discussed that the exact compounds present in the ESM taking place in the reduction might be amino acids proline (Yorseng etal. 2020) and/or hydroxyproline (Sinha and Ahmaruzzaman 2015). The latter contains two hydroxyl groups, which makes it a very probable candidate to take part in the reduction process. However, identifying theexact mechanism of Ag NPs formation using biological substances is a very complicated issue, because biological materials like plants are very complex and contain a large number of phytochemicals (4000 phytochemicals has been identified so far) (Das and Brar 2013). The obtained XRD data presented in Fig.1 were subjected to Rietveld refinement to determine the phase composition in percent and crystallite size of the produced Ag nanoparticles (this was estimated using Scherrer’s equation). The results are summarized in Table2. The results in Table2 are in accordance with the description of Fig.1 in terms of phase composition. The Ag0 crystallite size is significantly larger when the milling for 120min was performed (samples T3, T5 and T7), which confirms the better reaction progress in this case (at first smaller particles are formed which grow into larger ones during the prolonged milling). In sample T9, which contained the highest amount of AgNO3 and eggshell, the presence of a small amount of Ag2CO3 as a result of metathesis reaction between CaCO3 and AgNO3 most probably took place. Such a reaction as also reported in Sun etal. (2015). It was discovered in the past that the AgNO3:plant mixtures where higher amount of AgNO3 was introduced are prone to backward transformation (Kováčová etal. 2020; Baláž etal. 2020), all the as-received samples were washed with distilled water to avoid this process. Atomic absorption spectrometry The silver and calcium content in all as-received and washed solid samples were determined using AAS. The results for Ag are provided in Table3 and the ones for Ca are in Supplementary Information (TableS2). In the last column, the content of the element after washing (W) is divided by its content in the as-received (milled, M) sampleto get W/MAg ratio. This represents the stability of the prepared Ag NPs. Table 2 Phase composition of the as-received samples and approximate crystallite size of Ag0 nanoparticles calculated by Scherrer’s equation The residual of least-squares value (Rp) and a goodness of fit (GOF) values are also provided. a In this sample, also 3.2% of silver carbonate Ag2CO3 was detected Sample AgNO3 (%) CaCO3 (%) Ag0 (%) Ag0 crystallite size (nm) Rp (%) GOF Ag-ES-SER T1 Not detected 94.3 5.7 5.6 ± 0.9 5.03 12.13 Ag-ES-SER T2 Not detected 92.2 7.8 5.1 ± 0.8 5.28 10.73 Ag-ES-SER T3 Not detected 95.2 4.8 38.0 ± 3.6 5.18 9.79 Ag-ES-SER T4 Not detected 69.4 30.6 5.3 ± 0.7 5.19 12.30 Ag-ES-SER T5 Not detected 77.1 22.9 15.8 ± 0.6 5.69 13.88 Ag-ES-SER T6 Not detected 90.9 9.1 5.5 ± 0.8 4.48 7.61 Ag-ES-SER T7 45.0 28.1 26.9 23.1 ± 0.8 3.99 6.90 Ag-ES-SER T8 54.3 36.1 9.6 6.5 ± 0.9 4.38 7.01 Ag-ES-SER T9a44.5 43.8 8.5 8.1 ± 0.9 4.66 7.65 1904 Applied Nanoscience (2022) 12:1899–1916 1 3 We have introduced this parameter in our previous works (Kováčová etal. 2020; Baláž etal. 2021a). The higher the W/MAg value, the better the stability of the Ag NPs (lower amount of Ag is washed out). The theoretical content of both Ag and Ca is very close to the values determined in the as-received samples in all the cases. The theoretical content of Ag in the ES and SER should be close to zero, but these natural materials could contain some trace amounts due to adsorption from surrounding environment duringtheir growth. The AAS has shown this concentration to be lower than 0.005% for both cases.With regards to Ag content in the synthesized samples T1–T9 (Table3), itincreased after washingin 6 out of 9 cases (thus leading to a W/MAg ratio higher than 1). The lower values were evidenced for the samples T6–T8. For T7 and T8, ahigher amount of AgNO3 introduced into the reaction mixture is most probably responsible for that, as a significant amount is not converted to insoluble Ag0 or Ag2CO3 and is, thus, washed out. In the case of T6, the milling time of 15min is most probably not satisfactory for the efficient stabilization. On the other hand, the highest W/MAgvalues were found for T4 and T5 samples, where the milling time was 120min. The same time was used for T7 sample, however, in that case, the amount of AgNO3 was too high to get the W/MAg value higher than 1. Interestingly, the XRD pattern (Fig.1 and Table2) of this sample has shown much higher content of Ag0 than in the case of T8 and T9 samples. Despite this, the results from AAS are vice versa (i.e., the W/MAg value is higher for T8 and T9 samples). This means that in the case of T7, a significant amount of Ag0 nanoparticles was formed, but their stabilization was insufficient and some NPs were most probably washed out during the washing process. In T7, sample, the ES/SER ratio was low, i.e., the SER seems to be a great reducing, but not so good stabilizing agent. Eggshell seems to be the opposite. However, in our previous work (Kováčová etal. 2020), when the AgNO3:SER ratio was 1:10, the W/MAg value was almost 2.5. In current work, in the samples where we have the lowest AgNO3 content, the AgNO3:ES/SER ratio is 1:15.7 and despite this, a maximum value 1.47 for sample T5 was evidenced. This means that the synergy between ES and SER is not very good. Just for comparison, the W/MAg values detected for lavender/AgNO3 system were almost always below 1in (Baláž etal. 2021a). Thus, the used biological material is strongly interconnected with the Ag NPs stabilization. Regarding calcium (TableS2), the W/MCa ratio was in the range between 1.06 and 1.24 for all the samples (with the exception of T8, where it was significantly higher), so the content of Ca was slightly enriched during the washing process in most cases. This is in accordance with washing out of some water-soluble plant components and nonreacted AgNO3 and non-stabilized Ag NPs and maintaining CaCO3 in the solid, thus leading to a relative increase in Ca content. In the case of sample T8, a significant amount of silver was washed out because of high starting amount of AgNO3 and short milling time, leading in turn to more significant relative enrichment of Ca. To demonstrate the relationship between the dissolution of Ag and Ca, Fig. S1 (in the Supplementary file) was elaborated. The obtained points seem to be almost complete mirror images, i.e., the higher W/MAg, the lower W/MCa and vice versa. This phenomenon is the most significant for the sample T8. For the samples T1–T5 and for T9, W/MAg is higher than W/MCa. For the rest of the samples, the situation is inverted. More discussion will be provided when describing Taguchi results. Table 3 Concentration of silver in as-received and washed samples determined using atomic absorption spectrometry and the corresponding W/MAg ratios Sample Theoretical content before milling (%) As-received (M, %) Washed (W, %) W/MAg ratio Ag-ES-SER T1 3.53 3.1 4.1 1.32 Ag-ES-SER T2 3.53 3.15 4.06 1.29 Ag-ES-SER T3 3.53 3.64 4.45 1.22 Ag-ES-SER T4 10.58 10.64 14.41 1.35 Ag-ES-SER T5 10.58 10.6 15.61 1.47 Ag-ES-SER T6 10.58 10.81 9.82 0.91 Ag-ES-SER T7 31.75 30.79 24.47 0.79 Ag-ES-SER T8 31.75 33.29 23.23 0.70 Ag-ES-SER T9 31.75 33.07 39.09 1.18 Pure ES ~ 0 < 0.005 Pure SER ~ 0 < 0.005 1905Applied Nanoscience (2022) 12:1899–1916 1 3 X‑ray diffraction ofwashed powders The XRD patterns of the washed powders are presented in Fig.2. The XRD patterns of the washed samples T1–T7-W contain reflections of calcite, CaCO3 and those of elemental Ag0. The peaks’ intensities of these two phases change, but in general, the reflections corresponding to calcite are more intensive (with the exception of sample T7-W). The XRD patterns of the last two samples, T8-W and T9-W are slightly different and contain a significant amount of silver carbonate (the main diffraction is broad and located at around 33°). The presence of this phase is mainly visible in the sample T9-W and thus, the progress of the abovementioned metathesis reaction is enhanced in the presence of water. As mentioned in Apalangya etal. (2014), the eggshell-based CaCO3, when transformed to nanocrystalline range and becoming mesoporous, is prone to much easier dissolution and could, thus, supply Ca(II) ions for the reaction with non-reacted AgNO3. The ES/SER ratio was 3.0 for this sample, which means there was a significantly larger amount of CaCO3-containing ES. The presence of Ag2CO3 phase was not significant for the other two samples where the same ES/SER ratio was used (T6-W and T3-W). In these two cases, the amount of introduced AgNO3 was low, so there was not enough Ag(I) ions available for the reaction with the carbonate. Also the XRD data of the washed samples were subjected to Rietveld refinement to determine the phase composition in percent and also crystallite size of the produced Ag nanoparticles was estimated using Scherrer’s equation (Table4). The results in Table4 are in accordance with the discussion above. In all samples except T7-W the content of calcite was higher than that of Ag0. The amount of silver nanoparticles is significant mainly in the samples T4-W and T7-W. For thesample T9-W, the amount of precipitated Ag2CO3 is higher than both CaCO3 and Ag0. The crystallite size of Ag NPs for the samples T3-W, T5-W and T7-W is larger than 10nm, and in all the other cases, the crystallites are smaller. These three mentioned samples were obtained after milling for 120min. The largest crystallites were observed for T3-W sample, which is also due to the lowest amount of AgNO3 introduced. In the supplementary information, the comparison of phase content of Ag0 and CaCO3 between the as-received and the washed samples is provided (Fig. S2). The Ag0 content (Fig. S2a) is higher for all washed samples, however, the difference changes with regards to individual samples. The largest difference was observed for the samples T4 and T7, where the amount of Ag0 phase is the largest. The difference is more-or-less proportional, i.e., the larger the amount of Ag0, the larger the difference is. The only exception from this trend is the sample T1, however, this might be caused by an error in calculation, as the content of Ag0 phase is low. Fig. 2 XRD patterns of washed Ag-ES-SER T1–T9-W samples. At the bottom, the positions and intensities of identified crystallographic phases according to ICSD database are provided for comparison: calcite 166,364; silver 180,878; silver carbonate 93,988; silver nitrate 201,605 Table 4 Phase composition of the washed samples and approximate crystallite size of Ag0 nanoparticles calculated by Scherrer’s equation The residual of least-squares value (Rp) and a goodness of fit (GOF) values are also provided Sample Ag2CO3 (%) CaCO3 (%) Ag0 (%) Ag0 crystallite size Rp (%) GOF Ag-ES-SER T1-W Not detected 82.8 17.2 5.0 ± 0.7 5.99 16.21 Ag-ES-SER T2-W Not detected 71.9 8.8 5.4 ± 0.6 5.27 10.52 Ag-ES-SER T3-W Not detected 94.5 5.5 35.1 ± 2.9 5.57 10.72 Ag-ES-SER T4-W Not detected 51.5 48.5 4.9 ± 0.5 6.75 18.73 Ag-ES-SER T5-W not detected 74.0 26.0 15.8 ± 0.3 6.26 14.69 Ag-ES-SER T6-W Not detected 87.9 12.1 5.4 ± 0.7 5.50 10.78 Ag-ES-SER T7-W Not detected 45.5 54.5 19.2 ± 1.0 7.34 23.38 Ag-ES-SER T8-W 1.4 69.6 29.0 5.9 ± 0.1 6.42 18.90 Ag-ES-SER T9-W 45.5 41.3 13.2 11.6 ± 2.8 4.15 5.75 1906 Applied Nanoscience (2022) 12:1899–1916 1 3 With regards to CaCO3 content (Fig. S2b), the situation is inverted and in most cases, the CaCO3 content decreased after washing. The exceptions from this were observed for the samples T7 and T8, what is a result of washing out alarge amount of non-reacted silver nitrate. The comparison of Ag0 crystallite size for all samples before and after washing is provided in Fig. S3. Only minimum changes have been evidenced as a result of washing in the case of Ag0 crystallite size. In general, the nanoparticles very slightly decreased in size after washing, what might be caused by the escaping of non-stabilized Ag atoms from the surface of the nanoparticles, mainly so-called dangling Ag0 atoms could be washed out. Taguchi andANOVA analysis The selected results from both AAS and the Rietveld refinement of XRD patterns have been used as responses in Taguchi calculations whichpotentially allows us to find optimum experimental conditions to reach the best results. Namely, the W/MAg ratio, Ag content in washed samples from AAS and Ag crystallite size in the washed samples from Rietveld refinement data were used. All the time the “larger is better” approach was used for the calculations, as in all the cases, the higher values complement more advanced reaction progress. In the case of Ag content determined from AAS, the values for samples T8 and T9 correspond to Ag present in both Ag0 and Ag2CO3. Therefore, a re-calculation was necessary. To do this, the weight compositions from the Rietveld refinement reported in Table5 were used and the content of Ag determined by AAS was divided between Ag0 and Ag2CO3 accordingly. The calculated values of Ag contentcorresponding to Ag0 for T8 and T9 samples were 22.38% and 10.57%, respectively. One of the main advantages of the Taguchi method is that it usually allows the determination of the optimum parameters for the studied system without the need of performing the experiments using all combinations of parameters (Taguchi 2004). However, in our case, the obtained optimum parameters are quite heterogeneous and do not allow us to draw a solid conclusion (see TableS3 in the Supplementary file). For neither of the responses, the shortest milling time (15min) was found to be optimum, however, there is a combination of all the other values with this exception. Thus, it seems that the proposed system is very heterogeneous and there are many factors not included into the proposed calculations that influence the results. In future, it will be necessary to fix some of the parameters that were altered herein and investigate the other parameters one-by-one. In addition to Taguchi calculations, we have performed one-way analysis of variance (ANOVA) to find the contribution in percent and p value (i.e., statistical significance) of each investigated parameter. The contributions in percent of individual parameters to the corresponding factors can be seen in Fig.3. In two out of three cases, the most important parameter is the AgNO3 mass. For the Ag0 content determined from AAS (Fig.3a), it is also a statistically significant parameter (p value was 0.022). The ES/SER ratio was the second most important parameter for the Ag content (Fig.6a), whereas for the W/MAg ratio it was milling time (Fig.3b). The situation is different with Ag0 crystallite size, where the milling time was found to be the most important and statistically significant (p value = 0.017). In all the other cases, the studied parameters were not statistically significant (p value > 0.05). Comparative experiments undertheideal conditions withonlySER orES X‑ray diffraction According to the obtained results, it was quite difficult to select the optimum conditions. With regards to the Ag content in the washed samples determined by AAS, the experiment T9 seems to be the best one (39.09% Ag was determined in the washed sample, see Table3). However, from Table 5 Rietveld refinement results of the fitting of XRD patterns of T9-W sample and the corresponding analogues milled in the presence of pure ES or SER The residual of least-squares value (Rp) and a goodness of fit (GOF) values are also provided Sample Phase composition ICSD no. Weight % Crystallite size (nm) Rp (%) GOF T9-W Calcite, CaCO3 (hexagonal) 166,364 37.4 23.31 ± 0.00 3.95 5.59 Silver, Ag (cubic) 180,878 16.0 8.08 ± 5.20 Silver carbonate, Ag2CO3 (hexagonal) 93,988 46.6 9.98 ± 0.52 T9-pure ES-W Calcite, CaCO3 (hexagonal) 166,364 54.9 28.52 ± 0.00 7.35 2.78 Silver, Ag (cubic) 180,878 4.9 14.22 ± 0.05 Silver carbonate, Ag2CO3 (hexagonal) 93,988 40.2 10.96 ± 0.52 T9-pure SER-W Silver, Ag (cubic) 180,878 100 44.5 ± 0.00 6.78 3.40 1907Applied Nanoscience (2022) 12:1899–1916 1 3 the XRD pattern it is clear that a high amount of silver is in the form of Ag2CO3 and not in the form of silver nanoparticles. From the phase purity point of view, the best result seems to be the experiment T7, where only the diffractions corresponding to CaCO3 and Ag0 could be identified (Fig.2) and the Ag content determined by AAS is still relatively high (24.47%). Nevertheless, as the presence of Ag2CO3 in the case of the experiment T9 is a new discovery and its Fig. 3 Analysis of variance (ANOVA) results: Percentual contribution of different parameters on: a Ag content in the washed samples determined by AAS, b W/MAg ratio, c Ag0 crystallite size calculated from Rietveld refinement Fig. 4 XRD patterns of Ag-ES-SER-T9 (T9) sample and the ones milled in the presence of pure ES or SER: a as-received, b washed. At the bottom, the positions and the intensities of identified crystallographic phases according to ICSD database are provided: calcite 166,364; silver 180,878; silver carbonate 93,988; silver nitrate 201,605 1914 Applied Nanoscience (2022) 12:1899–1916 1 3 composite nanomaterials could be used in specific applications depending on the microorganism to be affected. This study has shown that mechanochemistry is a great tool to utilize natural resources and waste to synthesize nanocrystalline materials and opens new pathways for research in future, mainly suggesting that upon proper adjustment of milling conditions, it might be possible to control the Ag2CO3/Ag0 ratio in the product. Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s1320402202417-6. Acknowledgements This work was supported by the International Visgerad Fund, scholarship number 52010810 and would not be possible without the financial support of the Slovak Research and Development Agency under the contract no. APVV-18-0357 and that of TheMinistryof Education,Science, Research and Sport of theSlovakRepublic Grant Agency (project 2/0112/22). The support of COST Action CA18112 MechSustInd (http:// www. mechs ustind. eu), supported by the COST Association (European Cooperation in Science and Technology, http:// www. cost. eu) is also acknowledged. Optical density and respiration studies were funded by the National Science Centre, Poland (PRELUDIUM Project no. 2018/31/N/NZ1/03064, granted to Adrian Augustyniak). Adrian Augustyniak was also supported by the German Research Foundation (DFG) as part of the Research Training Group on Urban Water Interfaces (GRK 2032). 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