Antibacterial mechanism of Xiuyan jade mineral waste as a natural inorganic antibacterial agent

The Xiuyan jade waste exhibits prominent antibacterial properties after mechanical activation; these properties can be further optimized through low-temperature annealing. To elucidate its antibacterial mechanism, analysis methods involving metal-ion leaching monitoring and zeta potential analysis were used. Although the activated waste cannot be easily dissolved in water, Mg2+ and Ca2+ ions precipitated when the waste particles were dispersed in water. The particle surface showed different degrees of electronegativity, which could be the main reason for the antibacterial properties of the material. This study proved the importance of the surface electrical properties of activated waste particles on their properties by observing their adsorption capacity for methylene blue. At the annealing temperature of 270 °C, the Xiuyan jade waste exhibited the highest electronegativity and optimal antibacterial properties as well as the adsorption rate of methylene blue, verifying the significance of electronegativity of activated mineral waste powders on the antibacterial mechanism.


Introduction
Jade is a representative material with considerable significance in the history of oriental civilization.As such, there is a long history of jade mining and research [1].With the advancement of technology and the research on the spiritual and health benefits of jade, the large amounts of waste produced in the process of jade mining are gradually becoming a concern [2].Researchers are striving to modify and reuse various solid wastes to optimize environmental resources [3][4][5][6], and such research is in high demand globally [7][8][9][10].This study focused on the jade waste from the Xiuyan area, Liaoning province.Few studies have focused on the reuse of the Xiuyan jade waste [11].
The composition of jade and mineral waste varies depending on their origin; however, most materials contain elements such as Mg, Ca, and Si.Several studies [12] have attempted to extract metallic elements from minerals to compensate for the lack of metal resources.Further, other researchers [13] have focused on the modification of mineral waste for reuse.Studies on the geological features of the Xiuyan jade deposit indicate that it is found in the Mg-rich carbonate metamorphic strata, which contain magnesite-dolomite rock and rhombohedral magnesite [14].For minerals rich in Mg and Ca, high-temperature calcination can produce alkaline substances, such as MgO and CaO, to activate their properties for reuse [15,16].However, the precipitation of alkaline crystals can cause a considerably high pH level in the waste for building materials [17].Contrary to previous reports, the collected Xiuyan mineral waste powder exhibited remarkable antibacterial performance after mechanical activation with a considerably lower pH level than the limit value, thereby greatly enhancing its practicality.
Xiuyan jade mineral waste exhibits unprecedented antibacterial effects, particularly for naturally occurring minerals that have only undergone mechanical activation.This study thoroughly explored the antibacterial mechanism of this inorganic antibacterial agent, in contrast to most inorganic antibacterial agents that are chemically synthesized.According to their antibacterial mechanisms, these inorganic antibacterial agents can be divided into metal-ion/metal-oxide-based agents and photocatalytic agents.Typical examples of photocatalytic agents are ZnO and TiO 2 [18][19][20].These agents can destroy the structure of the bacteria directly through the energy generated by the electrons or electron holes produced after light irradiation or indirectly by producing reactive oxygen.Metal-based antibacterial agents mainly rely on metal ions or their oxides [21][22][23][24][25], such as Ag, Cu, and Mg, to destroy the cell wall, membrane, or contents, thereby rendering the cells inactive.The antibacterial mechanism of the metal-based agents is generally more complex, involving multiple antibacterial modes [26][27][28].For example, direct contact with metal nanoparticles or metal ions may damage the cell wall and membrane or release metal ions or small particles from the antibacterial agent may enter the cell contents and cause damage.In addition, in-cell environmental factors may induce the production of reactive oxygen, which is unfavorable for bacterial survival.The exploration of the antibacterial mechanism of Xiuyan jade mineral waste helps to understand its physicochemical properties and provide a scientific basis for expanding its application.As this natural inorganic antibacterial agent is a waste resource, offering unique economic and resource advantages is important in creating a green living environment.

Materials
The Xiuyan jade stone waste material used in this experiment is from Xiuyan, Anshan, Liaoning.Escherichia coli (ATCC25922) and Staphylococcus aureus (ATCC6538) used in the experiment were provided by the China General Microbiological Culture Collection Center.The nutrient agar and broth required for the experiment were produced by Beijing Luqiao Technology Co., Ltd The analytical-grade methylene blue used was purchased from China National Pharmaceutical Group Chemical Reagent Co., Ltd and used in the experiment.The deionized water was made in the laboratory.

Mechanical activation and calcination
The collected Xiuyan jade mine waste was subjected to physical crushing using a TR1114 vertical mill.The ground powder samples were then heat-treated at different temperatures in a box resistance furnace (Model sx-4-10) with an empty-furnace heating rate of 80 r min −1 .For each temperature sample, the furnace temperature was raised to the set temperature, held for 2 h, and then naturally cooled to indoor ambient temperature.The samples were numbered based on the calcination temperature, such as S0, S180, and S270.

Basic characterization
The analysis of the crystal properties was performed using the x-ray diffractometer (D8 Advance) with a scanning range of 20°-70°, a scanning rate of 3°(2θ)/min, and a Cu target Ka (wavelength of 0.15 nm) at room temperature.The particle size properties were measured and analyzed using a Malvern 3000 laser particle sizer.The zeta potential performance test equipment used was the Malvern Zetasizer Nano ZS ZEN 3600 from the UK, with testing conducted at room temperature and three cycles of testing.The bacterial interaction process was observed using a JEOL JEM-2100F transmission electron microscope with an acceleration voltage of 200 kV.Fourier transform infrared (FTIR) spectra were obtained using a Bruker FTIR spectrophotometer (FTIR-650).The sample powders were mixed with KBr (1%) and pressed into pellets under a pressure of 10 5 Pa.A thermogravimetry-differential scanning calorimeter was used for the thermogravimetric analysis.For nonisothermal treatment, the samples were heated in the range of 25 °C-1000 °C at 10 °C min −1 in N 2 atmosphere.

Metal-ion leaching and pH testing
A certain amount of powder sample was added to a 100-mL volumetric flask, filled to the graduation line with water and shaken well.Subsequently, the prepared sample solution was placed in a dual-frequency constanttemperature numerical control ultrasonic cleaner (model KQ-300GVDV) for 2 h for ultrasonic dispersion, followed by static settling for 24 h.The sample solution was then transferred to a centrifuge tube, ultrasonicated and centrifuged at 8000 r min −1 for 30 min, removed from the centrifuge tube after centrifugation, filtered the upper clear liquid through a 0.22-μm filter membrane, and transferred to a 15-mL sample tube.A pH meter (Mettler-Toledo S400) was used to measure the pH value of the solution, and metal-ion concentration analysis was conducted with an inductively coupled plasma optical emission spectrometer (ICP-OES) from Agilent Technologies.

Antibacterial performance testing
The powder antibacterial experiment follows the standard 'Evaluation Methods for Antibacterial and Bacteriostatic Effects of Daily Chemical Products' QB/T 2738-2012, using Staphylococcus aureus ATCC6538 and Escherichia coli ATCC25922 as the test bacteria.The strains are cultivated at 37 °C for 24 h in an incubator.The initial microbial concentration is of the order of 10 3 cfu ml −1 .
Each sample was tested in triplicate and the average of the three experiments was used as the result.The antibacterial rate was calculated using Formula (1): ẃhere I is the average bacterial count of the samples (cfu/mL) and II is the average bacterial count of the test sample (cfu/mL).

Adsorption properties of methylene blue
A certain amount of Xiuyan jade tailings powder was weighed and transferred into a stoppered conical flask.Subsequently, 100 ml of a methylene blue solution of a certain concentration was added, shaken well, and adsorbed by standing still.At different time points, the upper clear liquid was removed, centrifuged (2000 r min −1 for 2 min), and separated.The residual methylene blue concentration was measured and the adsorption quantity was calculated according to Formula (2): C 0 is the initial concentration of methylene blue (mg/L), C t is the equilibrium concentration of methylene blue after adsorption (mg/L), V is the volume of the methylene blue solution after adsorption (L), and M is the quantity of the sample added (mg).

Results and discussion
Xiuyan jade stone waste exhibits a complex composition, as shown in figure 1, comprising seven detectable mineral crystal structures in the decreasing order of dolomite, serpentine, calcite, talc, amphibole, magnesite, and quartz.The content of each mineral phase was obtained through the Jade 5.0 x-ray diffraction (XRD) analysis software for reference.
The crystal structure of the sample is almost identical to that of the raw material at annealing temperatures of 400 °C and lower until the two characteristic peaks corresponding to magnesite at 32.2°and 53.9°weaken considerably as the annealing temperature is increased to 500 °C.At annealing temperatures of 600 °C, the two magnesite peaks almost disappeared.When the annealing temperature reaches 700 °C, the characteristic peak of serpentine at 24.6°gradually disappears, whereas characteristic peaks of MgO at 43.0°and 62.0°appear.Further increasing the annealing temperature to 800 °C decreases the characteristic peaks of calcite at 41.2°, talc at 30.9°, and aragonite at 29.4°considerably, whereas characteristic peaks of CaO and MgO are observed, as shown in  From the above results, the key point for crystal phase changes during annealing is considered to be 500 °C.In particular, annealing at temperatures below this point does not result in considerable crystal phase changes.
The measured pH values exhibit the same trend, as shown in table 1. PH values of the jade wastes are directly influenced by the treating temperatures, and an obvious increase of pH values could indicate that the mineral compositions changed a lot at that temperature, i.e. the basic oxides start to show up.
Figure 3 shows the TG-DSC-DTG curves for the Xiuyan jade tailings after mechanical activation.Weight loss changed slightly at temperatures below 500 °C, which may be caused by the loss of absorbed water which was primarily absorbed in the cracks or adsorbed on the surfaces of the mineral crystals.Most of the absorbed water was lost during the mechanical activation process.Above 500 °C, the weight loss becomes intensive owing to the decomposition of dolomite.As shown in the DTG curve, the weight loss exhibits a sharp rise above 620 °C   and reaches a maximum at approximately 760 °C, which was consistent with previous reports [29] on the heatinduced phase change of dolomite.While the endothermic valley shown in the DSC curve also indicates that the phase change occurred in the range of 500 °C-800 °C, the dolomite began to decompose into MgO and Ca(CO 3 ) 2 when the temperature exceeded 500 °C, and Ca(CO 3 ) 2 further decomposed into CaO and CO 2 at 760 °C.The phase change of dolomite was nearly complete at approximately 800 °C.
In the preliminary antibacterial test analysis, there is a considerable difference in the antibacterial effect of the Xiuyan jade mine tailings under different annealing conditions with a sample concentration of 5 g L −1 and a bacteria interaction time of 2 h, as shown in figure 4.Although the antibacterial effect gradually increases with increasing annealing temperature, the key point is still considered to be 500 °C, which denotes the notable influence of the change in the crystal phase on the antibacterial effect.When the temperature reaches 700 °C and 800 °C, the optimal antibacterial effects are achieved by precipitation of alkaline oxides MgO and CaO.However, combined with the pH results, when the annealing temperature reaches 500 °C or above, the material is considered unsuitable as an eco-friendly building material because of the excessively high pH value [17].Therefore, only the jade wastes annealed in the temperature at 400 °C or below are expected to be further applied in building materials.
The changes in the crystal phase structure are attributed to the high calcite content in the jade mineral waste [30].When the annealing temperature is approximately 500 °C, the following reaction occurs: When the annealing temperature continues to increase to 800 °C, further decomposition occurs as follows: The different degrees of activation of the antibacterial properties of the jade waste are attributed to the MgO and CaO precipitation during the phase transition process [31].
As reported by previous studies, Mg 2+ ions play important roles in the antibacterial activities for Mg-based materials, even when the Mg 2+ ions exist in a non-acid or non-basic environment [32].However, for the samples annealed at high temperatures (>500 °C), the antibacterial effect from the released Mg 2+ might be ignored owing to the existance of basic metal oxides MgO and CaO in large amounts.Meanwhile, for the samples annealed below 500 °C, the antibacterial effect from Mg 2+ ions is relatively important.To further explore the effects of the relatively low activation temperatures (<500 °C) on the antibacterial properties of the jade waste, the antibacterial reduction rates of wastes treated by the two strains of E. coli (ATCC25922) and S. aureus (ATCC6538) were recorded.The contact times are 6 h and 24 h, respectively (figures 5(a) and (b), respectively).The antibacterial effect considerably improved with longer contact times.At activation temperature of 270 °C or higher, the antibacterial reduction rate considerably increased.After an interaction time of 24 h, the antibacterial rate could reach up to 99%.After activation at 270 °C, the overall antibacterial effect was remarkable against both types of bacteria.
The Xiuyan jade tailings comprise various natural minerals.During their natural formation process, various functional groups are formed on the surfaces and cracks of these mineral materials.After mechanical activation, the particle size is reduced as well as the surface energy and the unsaturated bonds on the surface are increased.The Fourier transform infrared analysis of the Xiuyan jade tailings powder materials reveals a strong OHstretching vibration peak at 3692 cm −1 , Si-O-Si stretching vibration peaks at 1081 and 966 cm −1 , and an Mg-O stretching vibration peak at 615 cm −1 , indicating the presence of a large number of active functional groups, such as OH-, Si-O-Si, and O-Si-O unsaturated bonds in the Xiuyan jade tailings, as shown in figure 6.
After mechanical activation, the surface activity of the tailing powder was further enhanced by imparting functional properties to the material [33].Through the analysis of the zeta potential performance, different annealing temperatures have some influence on the electronegativity on the surface of the powder particles.As shown in figure 7, the zeta potential of as-annealed tailings is approximately −15.5 mV, whereas that of the tailings annealed at 270 °C is approximately −18.1 mV.When the annealing temperature reaches 500 °C, the surface charge of the particles appears positive.Zeta potential is often used to measure the stability of molecules or particles dispersed in a solution; a higher absolute value of the potential increases the stability of the solution system.As the annealing temperature exceeds 600 °C, the stability of the solution system decreases with increasing annealing temperatures.Figure 6 shows the test result with 0.5 g of the test powder sample dissolved in 100 ml of water.The stability of the solution system is significantly enhanced as the amount of the test sample is reduced in the experiment.
As shown in figure 8, the zeta potential monitored after different amounts of the test powder were dissolved in 100 ml of water followed the same trend.With the discovery of the negative electronegativity of antibacterial materials and their antibacterial abilities [32], a highly negative electronegativity value of the particle surface may  facilitate the easier free movement of particles around the bacteria, causing contact damage to the bacteria or allow certain molecules or ions to penetrate the cell membrane and damage the bacteria.From the perspective of the zeta potential values, the sample annealed at 270 °C exhibits a certain advantage, indicating that the degree of influence of the particle surface charge on the antibacterial properties is remarkable.
In addition, several trends are observed in the metal-ion precipitation test of the jade tailings, which depend on the amount of the tested sample.As shown in table 2, the amounts of tested powder are 0.05, 0.10, and 20 g.As the amount of the test powder increases, the amount of Mg 2+ and Ca 2+ precipitation does not change equally.When test powder is increased from 0.05 g to 0.1 g, the precipitation of the Mg ions increases significantly for the samples annealed at temperatures lower than 500 °C, whereas the increase in the Ca-ion precipitation is relatively small.The precipitation of Mg ions does not change significantly with the annealing temperature, whereas the precipitation of Ca ions gradually increases as the annealing temperature is increased up to 500 °C, where the precipitation of Ca ions suddenly decreases.As the annealing temperature is continuously increased, the precipitation of Ca ions gradually increases.At the annealing temperature of 800 °C, the precipitation of Ca ions sharply increases.However, for samples annealed at 800 °C, the precipitation of Mg ions sharply decreases.When the amount of the test powder is increased to 20 g, the precipitation of Mg ions increases considerably.Meanwhile, the precipitation of Ca ions considerably decreases for the samples annealed at temperatures lower than 500 °C.At temperatures higher than 500 °C, the precipitation of Ca ions increases considerably, whereas that of Mg ions decreases considerably.The coexistence of Mg and Ca ions exhibits a competitive relationship, which is closely related to their solubility in the water of different crystal phases, such as CaMg(CO 3 ) 2 , CaCO 3 , Mg(OH) 2 , and Ca(OH) 2 .Based on the analysis of the zeta potential and metal-ion precipitation, jade tailings powder can release a certain amount of Mg 2+ and Ca 2+ ions in the water solution in case of the samples annealed below 500 °C without the influence of alkaline oxidants.Moreover, the particle surface remains in a stable negative state after releasing a considerable amount of positive ions.This phenomenon can considerably contribute to the antibacterial properties of the material [34].Although there is not a direct relationship between the released metal ions and the antibacterial ability, the Zeta potentials seems more influential to the antibacterial ability of the jade wastes.The reason for this might be that the released metal ions contribute partially to the surface charges.The free radicals on the surface of mineral crystal may contribute more to the surface charges, which are not measured in this study.The mechanical milling of the jade wastes always lead to more surface area of waste particles owing to the refinement during milling, so it sounds reasonable that lots of free radicals are produced.Possibly, the annealing temperature of 270 °C is critical for the surface state influenced by the absorbed water in the crystal of the jade wastes.
In addition to the reported antibacterial properties, a previous study on the adsorption of methylene blue by Xiuyan jade mine tailings [35] showed that the adsorption performance of such samples activated through calcination at 270 °C are slightly better than that of others.Similar to the antibacterial properties, ensuring the negative surface charge of the particles is crucial for the adsorption of methylene blue, which is a cationic dye. Figure 9 shows the variation of the adsorption amount of methylene blue over time for samples that have been soaked for 24 h and those that have not (S0 and S270).The results show that the adsorption effect of the unsoaked sample is significantly better than that of the soaked sample, which is related to the weakening of the negative surface charge of the particles after soaking.The result also shows that S270 has a slightly better adsorption performance than S0, regardless of the soaking process.The analysis of the adsorption effect of methylene blue confirms that the negative surface charge of the particles plays an important role in the performance of Xiuyan jade mine tailings, which further demonstrates their antibacterial mechanism.Overall, the Xiuyan jade tailings could be used as a type of natural antibacterial agent.Approximately 0.5-20 tons of jade tailings are produced for every 1 t of jade production [36].To date, the accumulation of jade tailings, a potential safety hazard, has caused a severe waste of land resources contributing to environmental pollution.Therefore, recycling of jade tailings is very crucial to address these problems.Xiuyan jade tailings, a type of Mgbased antibacterial agent, are friendly for human health.No secondary pollutants form during its recycling process, which includes only the mechanical milling and low-temperature heating process.More importantly, the entire process is simple and very economical.

Conclusion
This study investigated the antibacterial properties of the activated Xiuyan jade tailings.The tailings exhibited certain antibacterial properties after mechanical activation, which were further optimized through lowtemperature annealing.The optimal antibacterial performance was achieved after annealing at 270 °C.The economical and simple process is one of the important factors which make the jade waste worth being recycled.The recycle process maybe applied into other mineral wastes.
Based on the results, we found that when the Xiuyan jade waste was only mechanically crushed, the antibacterial characteristic of the crushed powders was obvious.With further calcination, the antibacterial properties of the waste improved.The mechanism depends somewhat on the mineral composition.For waste minerals calcined above 500 °C, basic oxides are the primary cause for the apoptosis of bacterial cells.For waste tailings calcined below 400 °C, only minerals without basic oxides are left.Therefore, the primary antibacterial mechanisms are related to the released metal ions and free radicals.We could not directly quantify these ions or radicals; nonetheless, we showed that there exists an obvious relationship between the surface charges and the antibacterial properties.Hence, we deduce a conclusion on the mechanism, i.e., development of surface charges through the release of metal ions and free radicals is primarily responsible for the loss of bacterial cells.
Accordingly, recycled Xiuyan jade tailings, as a kind of valuable natural and environmentally friendly antibacterial agents, are worthy to be explored.

figure 2 .
figure 2. As the annealing temperature is changed, both the crystal phases of amphibole and quartz slightly change.From the above results, the key point for crystal phase changes during annealing is considered to be 500 °C.In particular, annealing at temperatures below this point does not result in considerable crystal phase changes.The measured pH values exhibit the same trend, as shown in table1.PH values of the jade wastes are directly influenced by the treating temperatures, and an obvious increase of pH values could indicate that the mineral compositions changed a lot at that temperature, i.e. the basic oxides start to show up.Figure3shows the TG-DSC-DTG curves for the Xiuyan jade tailings after mechanical activation.Weight loss changed slightly at temperatures below 500 °C, which may be caused by the loss of absorbed water which was primarily absorbed in the cracks or adsorbed on the surfaces of the mineral crystals.Most of the absorbed water was lost during the mechanical activation process.Above 500 °C, the weight loss becomes intensive owing to the decomposition of dolomite.As shown in the DTG curve, the weight loss exhibits a sharp rise above 620 °C

Figure 2 .
Figure 2. XRD patterns of the Xiuyan jade waste annealed under different temperatures.

Figure 5 .
Figure 5. Antibacterial deduction of Xiuyan jade waste treated by (a) E. coli and (b) S. aureus.

Figure 7 .
Figure 7. Zeta potential values of Xiuyan jade waste annealed at different temperatures.

Figure 8 .
Figure 8. Relationship between the Zeta potential values and the amount of jade waste powder (sample S270).

Figure 9 .
Figure 9. Relationship of the adsorption capacity of methylene blue with time.

Table 1 .
pH values of the Xiuyan jade waste before and after annealing.

Table 2 .
Amount of released metal ions.